Chemoselective thiol-conjugation with alkene or alkyne-phosphonamidates

ABSTRACT

Disclosed are novel conjugates and processes for the preparation thereof. A process for the preparation of alkene- or alkyne-phosphonamidates comprises the steps of
         (I) reacting a compound of formula (III)       

     
       
         
         
             
             
         
       
         
         
           
             
               
                 with an azide of formula (IV) 
               
             
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 to prepare a compound of formula (V) 
               
             
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             (II) reacting a compound of formula (V) with a thiol-containing molecule of formula (VI) 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 resulting in a compound of formula (VII)

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 16/328,199, filed Feb. 25, 2019, which is a U.S. national phaseapplication of International Application No. PCT/EP2017/071937, filedSep. 1, 2017, which claims priority to European Patent Application No.16001917.0, filed Sep. 1, 2016. U.S. patent application Ser. No.16/328,199 is hereby incorporated in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 350066_401D1_SEQUENCE_LISTING.txt. The text fileis 5 KB, was created on Aug. 28, 2021, and is being submittedelectronically via EFS-Web.

BACKGROUND

Chemoselective and bioorthogonal reactions have emerged as powerfultools for the site-specific modification of proteins (1, 2). With thesereactions, various protein- and antibody-conjugates became accessible,which carried functional modules like fluorophores and otherspectroscopic labels, polymers, toxins as well as small molecules andproteins that resemble posttranslational protein modifications. Thereby,chemoselective protein modification techniques have greatly contributedto fundamental studies ranging from the investigation of biologicalfunctions of proteins and the development of new imaging techniques topromising new medicinal approaches in diagnostics, the design ofprotein-based pharmaceuticals and the targeted-delivery of drugs.

Over the last years, researchers have mainly concentrated on twodifferent aspects in the engineering of bioorthogonal reactions for themodification of proteins (3). On the one hand, many efforts have beendevoted to fast reactions requiring highly reactive starting materialsfor the transformation of unique functionalities present in proteinside-chains (4, 5). This approach is complimented by advanced ambersuppression techniques to achieve a site-specific labeling, whichresulted in a number of genetically encoded, highly reactivebioorthogonal reporters to undergo various types of cycloadditionreactions, including strain-promoted alkyne-azide cycloaddition orinverse-demand Diels-Alder reactions (6, 7). On the other hand,researchers have focused on developing and applying high-yieldingprotein modification reactions, especially if high amounts of functionalprotein-conjugates and ideally quantitative conversions are desired toavoid tedious if not impossible purification steps (1). To achieve this,high yields in protein expression are of particular importance. Sinceamber suppression can result in low amounts of expressed protein,standard and auxotrophic expression systems are often preferred. Acommon scenario to achieve site-specific labeling in combination withstandard protein expression is the placement of a unique Cys residue ina protein of choice by site-directed mutagenesis, followed byCys-modification strategies (8). Alternatively, azide- oralkyne-containing amino acids can be incorporated using auxotrophicexpression systems (9), which can be modified using Staudinger ligationsand Cu-catalyzed azide-alkyne cycloaddition (CuAAC) (10, 11).

While both of these aspects have seen significant advancements in recentyears, a general and modular accessibility of highly reactive andcomplex functional modules for a metal-free chemoselective modificationreaction remains often challenging. This is due to the requirement ofadditional protecting group manipulations in the synthesis of reactivebioorthogonal building blocks, which can be problematic in light of thehigh reactivity and lability of the employed bioorthogonal functions.For example, the synthesis of a highly reactive cyclooctyne-containingfluorescent peptide carrying a Xe-cryptophane for molecular imaging,required a sophisticated yet low yielding use of orthogonal protectinggroups (12).

In 2013, a modular chemoselective method for the stepwise coupling oftwo azide-building blocks by combining a CuAAC with theStaudinger-phosphonite reaction (SPhR) was developed (13). Introductionof SPhR for the chemoselective labeling of azido-containing peptides andproteins in aqueous systems to form protein-phosphonamidate-conjugatesvia boran protected bisethoxyalkylene-phosphonite is known from (14).

Previous techniques for the conjugation of Cys residues rely mainly onmaleimide conjugation, which often tend to hydrolyze and are prone tothiol exchange under high thiol concentrations. For a recentcomprehensive overview on Cys-conjugation techniques (22). WO2015/169784 discloses a process for the preparation ofC2-disulfide-bridged peptides and proteins, wherein the bridging isachieved by a thiol-yn-reaction with alkynes. U.S. Pat. No. 2,535,174describes the alkaline catalyzed addition of saturated aliphaticmercaptans to esters of ethenephosphonic acids. However, thethiol-conjugates of alkyne and alkene-phosphonamidates as disclosedherein have neither been reported nor are they anticipated by the priorart.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the hydrolytic decay of a glutathion-phosphonamidateconjugate according to Scheme 5 under acidic conditions.

FIG. 2 shows the conversion of an alkynylphosphonamidate to the thioladduct, determined by HPLC.

FIG. 3 shows a LC/MS analysis of the addition of glutathione (GSH) tocyclic c(RGDfK)-phosphonamidate alkyne

to give

FIG. 4 shows the pH-dependent stability of c(RGDfK)-Glutathion

FIG. 5 shows the Staudinger-induced thiol-addition to a singleaddressable Cys-containing eGFP, in which the conjugate depicted in FIG.30 was formed again in quantitative conversion after 3 h incubation at37° C.

FIG. 6 shows a ³¹P NMR-NMR from the crude synthesis ofbisethoxyalkyne-phosphonite

where the full consumption of the starting material was confirmed(product at 126.73 ppm).

FIG. 7 shows the UPLC chromatogram of purified c(RGDfK)-azide

FIG. 8 shows the LC-UV chromatogram of purified c(RGDfK)-alkyne

FIG. 9 shows the LC-UV chromatogram of purified c(RGDfK)-glutathion

FIG. 10 shows a western blot analysis after non reducing SDS-PAGE. 1:Cetuximab starting material. 2: 5 min, 3: 1 h, 4: 2 h, 5: 20 hincubation with a biotin modified phosphonamidate. Reaction with (left)and without (right) prior reduction of the disulfides.

FIG. 11 shows the stability of ethyl-N-phenyl-P-vinyl-phosphonamidate todifferent pHs over time. Stability of the phosphonamidate to differentpHs was proven by ³¹P-NMR in aqueous buffers at room temperature.

FIG. 12 shows the stability of a glutathione phosphonamidate adduct todifferent pHs over time. Stability of a thiol-adduct to different pHswas proven by ³¹P-NMR in aqueous buffers at room temperature.

FIG. 13 shows the consumption of various N-phenyl vinyl phosphonamidatesin the reaction with glutathione at pH 8.5. HPLC UV traces were taken atdifferent time points. Experiments were performed in triplicates.

FIG. 14 shows a western blot analysis after non reducing SDS-PAGE. 1:Cetuximab starting material. 2: 5 min, 3: 1 h, 4: 2 h, 5: 20 hincubation with a biotin modified phosphonamidate. Reaction with (left)and without (right) prior reduction of the disulfides.

FIG. 15 shows the sequences mentioned throughout the specification.

FIG. 16A shows trastuzumab modification with three differentCys-reactive biotin derivatives.

FIG. 16B shows the western blot analysis. Lanes 1 and 5: untreatedantibody. Lanes 2-4: reactions with prior DTT treatment. Lanes 6-8:Control reactions without prior DTT treatment.

FIGS. 17A to 17C show trastuzumab modification with phosphonamidatemodified, cathepsin cleavable MMAF (Amidate-VC-PAB-MMAF). FIG. 17A showsthe reaction scheme comprising reduction and alkylation of interchaindisulfides. FIG. 17B shows SDS-PAGE analysis of the reaction. FIG. 17Cshows the deconvoluted MS spectra of the antibody fragments afterdeglycosylation with PNGase F and reduction with DTT. LC: Light chain;HC: Heavy chain; mod: Amidate-VC-PAB-MMAF.

FIG. 18 shows the increased antiproliferative potency of MMAF linkedtrastuzumab on two different Her2 overexpressing cell lines (BT474 andSKBR3) and one control (MDAMB468). Plots depict the number ofproliferating cells after 4 days of antibody treatment in dependency ofthe antibody concentration. Trastuzumab alone,trastuzumab-phosphonamidate-MMAF and trastuzumab-maleimide-MMAF weretested.

FIG. 19 shows the UPLC-UV chromatogram of purifiedphosphonamidate-Val-Cit-Pab-MMAF

FIG. 20 shows immunostainings of fixed cells over expressing the cellsurface receptor Her2 (BT474 and SKBR3) or exhibiting low Her2expression levels (MDAMB468).

FIG. 21A shows the structure of a phosphonamidate linked FRET conjugate.FIG. 21B shows the structure of a maleimide linked FRET conjugate. FIG.21C shows the principle of the fluorescence-quencher based readout. FIG.21D shows monitoring of the fluorescence increase over time. FIG. 21Eshows the comparison of a phosphonamidate- and a maleimide-linkeddye-quencher pair during exposure to 1000 eq. glutathion in PBS.

FIGS. 22A and 22B show the transfer of the antibody modification toserum proteins. FIG. 22A shows incubation of Tratuzumab-biotinconjugates. FIG. 22B shows monitoring of the biotin transfer to albuminby western blot analysis. Lane 1: Untreated maleimide conjugate. Lanes2-5: Analysis of the BSA exposed maleimide adduct after 0, 1, 2 and 5days. Lane 6: Untreated amidate conjugate. Lanes 7-10: Analysis of theBSA exposed amidate adduct after 0, 1, 2 and 5 days.

FIGS. 23A to 23D show the determination of the second order rateconstant of the thiol addition. FIG. 23A shows the reaction of the EDANSphosphonamidate with glutathione. FIG. 23B shows the fluorescence HPLCtrace after 30 min reaction time. FIG. 23C shows the monitoring of thephosphonamidate decrease over time. FIG. 23D shows the plot of theinverse concentration against reaction time. Error bars represent themean of three replicates (n=3).

FIG. 24 shows the reaction of an azido modified peptide with the watersoluble phosphonite E1 in Tris buffer. The upper portion shows thereaction scheme. The lower portion shows the HPLC-trace, startingmaterial and reaction after 2 h were tested.

FIG. 25A shows a HPLC trace of the first conjugate according to Table 5.FIG. 25B shows a HPLC trace of the second conjugate according to Table5. FIG. 25C shows a HPLC trace of the third conjugate according to Table5. FIG. 25D shows a HPLC trace of the fourth conjugate according toTable 5. FIG. 25E shows a HPLC trace of the fifth conjugate according toTable 5.

FIG. 26A to 26C show the UPLC-MS analysis of the cleavage of disulfidecontaining amidate-adducts SM1, SM2 and SM3 with TCEP. Incubation withTCEP, and with PBS only were tested. Peaks were identified by MS. FIG.26A shows the UPLC-MS analysis for SM1.

FIG. 26B shows the UPLC-MS analysis for SM2. FIG. 26C shows the UPLC-MSanalysis for SM3.

FIG. 27 shows the UPLC-MS analysis of the cleavage of theester-containing amidate-adduct SM4 with cell lysate. Incubation withcell lysate and with PBS only were tested. Peaks were identified by MS.

FIG. 28 shows the cleavage of the diazo-containing amidate-adduct SM5with sodium dithionite. Incubation with TCEP and with PBS only weretested. Peaks were identified by MS.

FIG. 29 shows the thiol addition of alkyne-c(Tat) to eGFP C70M S147C.

FIG. 30 shows a conjugate of formula (VII), which comprises a GFPprotein, preferably an eGFP protein, on the left side and a cyclid RGDpeptide on the right side.

FIG. 31 shows an addition of a water-soluble vinyl phosphonamidate toeGFP with one addressable cysteine to form a conjugate of formula (VII).

FIG. 32 shows a Cy5 phosphonamidate labeling of eGFP with oneaddressable cysteine to form a conjugate of formula (VII). In gelfluorescence read out after SDS Page confirms selective Cy5 labeling.

FIG. 33 shows a photocleavable alkyne labeling of eGFP with oneaddressable cysteine to form a conjugate of formula (VII), subsequentbiotin labeling via CuACC, and western blot analysis.

FIG. 34 shows a photocleavable alkyne labeling of ubiquitin with oneaddressable cysteine to form a conjugate of formula (VII), andsubsequent biotin labeling via CuACC. Western blot analysis afterimmobilization on streptavidin beads. 1: ubiquitin starting material, 2:reaction mixture after CuACC, 3: supernatant after incubation of thereaction mixture with streptavidin agarose, 4: flow through after washof streptavidin agarose, 5: boiled beads, 6: irradiated beads.

FIG. 35 shows the principle of alkyne-phosphonamidate-synthesis andsubsequent chemoselective modification of Cys-residues.

FIG. 36 describes the general strategy for a synthesis according to thepresent invention at the example of ethenyl or ethynyl phosphonites. R1represents an optionally substituted aliphatic or aromatic residue.

FIG. 37 shows the difference between a process known in the art (e.g.,15) and a process according to the present invention. FIG. 37A showssequential azide-azide couplings using alkyne phosphonites. FIG. 37Bshows a Staudinger-induced thiol-addition (the thiol addition may bealso denoted as “Michael addition”) for the modification of Cys residuesaccording to the invention. Merely as examples, ethenyl and ethynyl(diethyl)phosphonite were used.

FIG. 38 shows that the incorporation of both an azide and a thiol intothe same molecule provides for an intramolecular Staudinger-inducedthiol addition that can realize an intramolecular cyclization.

FIG. 39 shows a fluorescently labeled ASGP-R addressing Cys conjugate offormula (X) which can be produced via the modular addition to vinylphosphonamidates.

FIG. 40 shows the Staudinger-induced thiol-addition of cyclicazido-RGD-peptides to GSH.

FIG. 41 shows the two-step reduction and alkylation approach forcysteine selective antibody modification with a biotin modified alkynylphosphonamidate.

FIG. 42 shows the reduction of antibody disulfides and subsequentmodification with a biotin vinyl phosphonamidate.

FIG. 43 shows the synthesis of a fluorescently labeled ASGP-R addressingCy5 conjugate via the modular addition to vinyl phosphonamidates.

FIG. 44 shows the general procedure for the modification of Trastuzumabvia the reduction/alkylation protocol.

FIG. 45 shows procedures for the Staudinger-induced thiol addition withalkynyl-phosphonites for the generation of Antibody FluorophoreConjugates (AFCs).

FIG. 46 shows the synthesis of a conjugate having a cleavabledisulfide-comprising O-substituent via the Staudinger phosphonitereaction, followed by the addition of a thiol-containing molecule to theunsaturated phosphonamidate.

FIG. 47 shows the general procedure 5 for the addition of a Cys-modelpeptide to different 0-Substituted EDANS phosphonamidates.

FIG. 48 shows the procedure for the cleavage of the disulfide containingamidate-adducts with TCEP.

FIG. 49 shows the procedure for the cleavage of the ester-containingamidate-adducts with cell lysate.

FIG. 50 shows the procedure for the diazo-containing amidate-adductswith sodium dithionite.

FIG. 51 shows the reductive cleavage and elimination mechanism.

FIG. 52 shows the SPPS of alkyne functionalized cyclic Tat.

FIG. 53 shows the hydrothiolation reaction of an electron-deficientc(Tat)-phosphonamidate alkyne with GFP C70M S147C.

FIG. 54 shows the BCL9-azide.

FIG. 55 shows the Staudinger Reaction on BCL9-azide withalkyne-phosphonamidate.

FIG. 56 shows the Staudinger Reaction on BCL9-azide withalkene-phosphonamidate.

DEFINITIONS

The person skilled in the art is aware that the terms “a” or “an”, asused in the present application, may, depending on the situation, mean“one (1)” “one (1) or more” or “at least one (1)”.

Halogen, unless defined otherwise: elements of the 7^(th) main group,preferably fluorine, chlorine, bromine and iodine, more preferablyfluorine, chlorine and bromine and, in combination with Mg even morepreferably bromine.

alkyl, unless defined otherwise elsewhere: saturated straight-chain orbranched hydrocarbon radicals having preferably (C₁-C₈)-, (C₁-C₆)- or(C₁-C₄)-carbon atoms. Examples: methyl, ethyl, propyl, 1-methylethyl,butyl, etc.

Alkenyl, unless defined otherwise elsewhere: unsaturated straight-chainor branched hydrocarbon radicals having a double bond. Alkenyl ispreferably (C₂-C₈)-, (C₂-C₆)- or (C₂-C₄)-alkenyl. Examples: ethenyl,1-propenyl, 3-butenyl, etc.

Alkynyl, unless defined otherwise elsewhere: unsaturated straight-chainor branched hydrocarbon radicals having a triple bond. Alkynyl ispreferably (C₂-C₈)—, (C₂-C₆)- or (C₂-C₄)-alkynyl. Examples: ethynyl,1-propynyl, etc.

Alkoxy (alkyl radical —O—), unless defined otherwise elsewhere: an alkylradical which is attached via an oxygen atom (—O—) to the basicstructure. Alkoxy is preferably (C₁-C₈)-, (C₁-C₆)- or (C₁-C₄)-alkoxy.Examples: methoxy, ethoxy, propoxy, 1-methylethoxy, etc.

Analogously, alkenoxy and alkynoxy, unless defined otherwise elsewhere,are alkenyl radicals and alkynyl radicals, respectively, which areattached via —O— to the basic structure. Alkenoxy is preferably(C₂-C₈)—, (C₂-C₆)- or (C₂-C₄)-alkenoxy. Alkynoxy is preferably(C₃-C₁₀)-, (C₃-C₈)- or (C₃-C₄)-alkynoxy.

alkylcarbonyl (alkyl radical —C(═O)—), unless defined otherwise:alkylcarbonyl is preferably (C₁-C₈)-, (C₁-C₆)- or (C₁-C₄)-alkylcarbonyl.Here, the number of carbon atoms refers to the alkyl radical in thealkylcarbonyl group.

Analogously, alkenylcarbonyl and alkynylcarbonyl, are, unless definedotherwise elsewhere: alkenyl radicals and alkynyl radicals,respectively, which are attached via —C(═O)— to the basic structure.Alkenylcarbonyl is preferably (C₂-C₈)-, (C₂-C₆)- or(C₂-C₄)-alkenylcarbonyl. Alkynylcarbonyl is preferably (C₂-C₈)—,(C₂-C₆)- or (C₂-C₄)-alkynylcarbonyl.

Alkoxycarbonyl (alkyl radical —O—C(═O)—), unless defined otherwiseelsewhere: alkoxycarbonyl is preferably (C₁-C₈)-, (C₁-C₈)- or(C₁-C₄)-alkoxycarbonyl. Here, the number of carbon atoms refers to thealkyl radical in the alkoxycarbonyl group.

Analogously, alkenoxycarbonyl and alkynoxycarbonyl, unless definedotherwise elsewhere, are: alkenyl radicals and alkynyl radicals,respectively, which are attached via —O—C(═O)— to the basic structure.Alkenoxycarbonyl is preferably (C₂-C₈)—, (C₂-C₆)- or(C₂-C₄)-alkenoxycarbonyl. Alkynoxycarbonyl is preferably (C₃-C₈)-,(C₃-C₆)- or (C₃-C₄)-alkynoxycarbonyl.

alkylcarbonyloxy (alkyl radical —C(═O)—O—), unless defined otherwiseelsewhere: an alkyl radical which is attached via a carbonyloxy group(—C(═O)—O—) by the oxygen to the basic structure. alkylcarbonyloxy ispreferably (C₁-C₈)-, (C₁-C₆)- or (C₁-C₄)-alkylcarbonyloxy.

Analogously, alkenylcarbonyloxy and alkynylcarbonyloxy, unless definedotherwise elsewhere, are: alkenyl radicals and alkynyl radicals,respectively, which are attached via (—C(═O)—O—) to the basic structure.Alkenylcarbonyloxy is preferably (C₂-C₈)—, (C₂-C₆)- or(C₂—C₄)-alkenylcarbonyloxy. Alkynylcarbonyloxy is preferably (C₂-C₈)-,(C₂—C)- or (C₂-C₄)-alkynylcarbonyloxy.

alkylthio, unless defined otherwise elsewhere: an alkyl radical which isattached via —S— to the basic structure. alkylthio is preferably(C₁-C₈)-, (C₁-C₆)- or (C₁-C₄)-alkylthio.

Analogously, alkenylthio and alkynylthio, unless defined otherwiseelsewhere, are: alkenyl radicals and alkynyl radicals, respectively,which are attached via —S— to the basic structure. Alkenylthio ispreferably (C₂-C₈)-, (C₂-C₆)- or (C₂-C₄)-alkenylthio. Alkynylthio ispreferably (C₃-C₈)-, (C₃-C₆)- or (C₃-C₄)-alkynylthio.

The term “substituted” as used unless defined otherwise elsewhere,refers to a very broad substitution pattern. As can be seen from thedisclosure of this invention, especially position R₁,

and ● allow the substitution with numerous organic (macro)molecules. Itis submitted that the structure of these molecules is not relevant forthe presently disclosed process and the resulting conjugates. Thus, itwould represent an undue limitation to limit the principle of this newand innovative concept to only some molecules. Nevertheless, it issubmitted that the term refers to organic substituents or salts thereof,respectively, which may again be substituted several times by furtherorganic substituents or salts thereof, respectively. Examples for suchcomplex substituents were produced and are presented in this application(see, e.g. Schemes 5, 6, 7, 11, 13, 15, 19, 20, 21, 22, 23, and 24).Preferably, the term substituted refers to groups which are substitutedwith one or more substitutents selected from nitro, cyano, Cl, F, Cl,Br, —NH—R, NR₂, COOH, —COOR, —OC(O)R—NH₂, —OH, —CONH₂CONHR, CON(R)₂,—S—R, —SH, —C(O)H, —C(O)R, (C₁-C₂₀)-alkyl, (C₁-C₂₀)-alkoxy,(C₂-C₂₀)-allyl, (hetero)cyclic rings of 3 to 8 ring-members wherein, ifpresent, the heteroatom or atoms are independently selected from N, Oand S, (hetero)aromatic systems with 5 to 12 ring atoms (e.g., phenyl,pyridyl, naphtyl etc.), wherein R again can represent any of thesesubstituents and the substitution can be repeated several times, forexample, substitution can be repeated for 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 times; see, e.g. the ● substituent in Scheme 11:

wherein # represents the position of N₃ or N if ● is already part of acompound of formula (VII). However, the skilled person will agree thatan alkyl-chain which is substituted with a polysaccharide of 40 unitscannot be simply described by general substitution pattern.

The terms “peptide” as used herein refers to an organic compoundcomprising two or more amino acids covalently joined by peptide bonds(amide bond). Peptides may be referred to with respect to the number ofconstituent amino acids, i.e., a dipeptide contains two amino acidresidues, a tripeptide contains three, etc. Peptides containing ten orfewer amino acids may be referred to as oligopeptides, while those withmore than ten amino acid residues are polypeptides. The amino acids canform at least one circle or a branched or unbranched chain or mixturesthereof. Proteins and antibodies are peptides and, thus, encompassed bythe term, but may be named separately, due to their importance.

The term “amino acid” as used herein refers to an organic compoundhaving a —CH(NH₃)—COOH group. In one embodiment, the term “amino acid”refers to a natural occurring amino acid arginine, lysine, asparticacid, glutamic acid, glutamine, asparagine, histidine, serine,threonine, tyrosine, cysteine, methionine, tryptophan, alanine,isoleucine, leicine, phenylalanine, valine, proline and glycine.However, the term in its broader meaning also encompasses non-naturaloccurring amino acids.

Amino acids and peptides according to the invention can also be modifiedat functional groups. Non limiting examples are saccharides, e.g.,N-Acetylgalactosamine (GalNAc), or protecting groups, e.g.,Fluorenylmethoxycarbonyl (Fmoc)-modifications or esters.

The term “protein” refers to peptides which comprise one or more longchains of amino acid residues. Proteins perform a vast array offunctions in vivo and in vitro including catalysing metabolic reactions,DNA replication, responding to stimuli, and transporting molecules,catalysing reactions. Proteins are folded into a specificthree-dimensional structure. The residues in a protein are oftenchemically modified, e.g., by post-translational modification, whichalters the physical and chemical properties, folding, stability,activity, and ultimately, the function of the proteins. Sometimesproteins have non-peptide groups attached, which can be calledprosthetic groups or cofactors. Proteins, including enzymes andcoenzymes, can also work together to achieve a particular function, andthey often associate to form stable protein complexes. All these formsare encompassed by the term “protein”.

The term “protein tags” as used herein refers to peptide sequences whichcan be attached to proteins or other thiol-comprising compounds via thelinker according to the present invention for various purposes. Nonlimiting examples for protein tags are affinity tags, solubilizationtags, chromatography tags epitope tags and reporter enzymes.

Affinity tags are appended to proteins and other thiol-comprisingcompounds via the linker according to the present invention so that theycan be, e.g., purified using an affinity technique. These include forexample chitin binding protein (CBP), maltose binding protein (MBP), andglutathione-S-transferase (GST) or the poly(His) tag.

Solubilization tags can be used to assist in the proper folding inproteins and keep them from precipitating. These include thioredoxin(TRX) and poly(NANP). Some affinity tags have a dual role as asolubilization agent, such as MBP, and GST.

Chromatography tags are used to alter chromatographic properties of theprotein to afford different resolution across a particular separationtechnique. Often, these consist of polyanionic amino acids, such asFLAG-tag.

Epitope tags are short peptide sequences which are chosen becausehigh-affinity antibodies can be reliably produced in many differentspecies. These are usually derived from viral genes. Epitope tagsinclude V5-tag, Myc-tag, HA-tag and NE-tag. These tags are particularlyuseful for western blotting, immunofluorescence and immunoprecipitationexperiments, and antibody purification.

The term “reporter enzymes” as used herein refer to any known enzymewhich allows an increase of a signal in a biochemical detection. Nonlimiting examples are, colorant forming enzymes such as alkalinephosphatase (AP), horseradish peroxidase (HRP) or glucose oxidase (GOX);fluorescent proteins, such as green fluorescence protein (GFP), redoxsensitive GFP (RoGFP), Azurite or Emerald; luciferase, i.e. a class ofoxidative enzymes that produce bioluminescence (e.g. firefly luciferase(EC 1.13.12.7)); chloramphenicol acetyl transferase (CAT);ß-galactosidase; or ß-glucuronidase.

Non-limiting examples of protein tags are: AviTag, a peptide allowingbiotinylation by the enzyme BirA and so the protein can be isolated bystreptavidin (GLNDIFEAQKIEWHE), Calmodulin-tag, a peptide bound by theprotein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL), polyglutamate tag, apeptide binding efficiently to anion-exchange resin such as Mono-Q(EEEEEE), E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR),FLAG-tag, a peptide recognized by an antibody (DYKDDDDK), HA-tag, apeptide from hemagglutinin recognized by an antibody (YPYDVPDYA)His-tag, 5-10 histidines bound by a nickel or cobalt chelate (HHHHHH),Myc-tag, a peptide derived from c-myc recognized by an antibody(EQKLISEEDL), NE-tag, a novel 18-amino-acid synthetic peptide(TKENPRSNQEESYDDNES) recognized by a monoclonal IgG1 antibody, which isuseful in a wide spectrum of applications including Western blotting,ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, andaffinity purification of recombinant proteins, S-tag, a peptide derivedfrom Ribonuclease A (KETAAAKFERQHMDS), SBP-tag, a peptide which binds tostreptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), Softag 1, formammalian expression (SLAELLNAGLGGS), Softag 3, for prokaryoticexpression (TQDPSRVG), Strep-tag, a peptide which binds to streptavidinor the modified streptavidin called streptactin (Strep-tag II:WSHPQFEK), TC tag, a tetracysteine tag that is recognized by FlAsH andReAsH biarsenical compounds (CCPGCC), V5 tag, a peptide recognized by anantibody (GKPIPNPLLGLDST), VSV-tag, a peptide recognized by an antibody(YTDIEMNRLGK), Xpress tag (DLYDDDDK), Isopeptag, a peptide which bindscovalently to pilin-C protein (TDKDMTITFTNKKDAE), SpyTag, a peptidewhich binds covalently to SpyCatcher protein (AHIVMVDAYKPTK), SnoopTag,a peptide which binds covalently to SnoopCatcher protein (KLGDIEFIKVNK),BCCP (Biotin Carboxyl Carrier Protein), a protein domain biotinylated byBirA enabling recognition by streptavidin,Glutathione-S-transferase-tag, a protein which binds to immobilizedglutathione, Green fluorescent protein-tag, a protein which isspontaneously fluorescent and can be bound by nanobodies, Halo-tag, amutated hydrolase that covalently attaches to the HaloLink™ Resin(Promega), Maltose binding protein-tag, a protein which binds to amyloseagarose, Nus-tag, Thioredoxin-tag, Fc-tag, derived from immunoglobulinFc domain, allow dimerization and solubilization. Can be used forpurification on Protein-A Sepharose, Designed Intrinsically Disorderedtags containing disorder promoting amino acids (P, E, S, T, A, Q, G, . .. ), alkaline phosphatase (AP), horseradish peroxidase (HRP) glucoseoxidase (GOX), green fluorescence protein (GFP), redox sensitive GFP(RoGFP), Azurite, Emerald, firefly luciferase (EC 1.13.12.7)),chloramphenicol acetyl transferase (CAT), ß-galactosidase,ß-glucuronidase, tubulin-tyrosine ligase (TTL).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules, preferably comprised of four polypeptidechains, two heavy (H) chains and two light (L) chains which aretypically inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region cancomprise e.g. three domains CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain (CL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is typicallycomposed of three CDRs and up to four FRs arranged from amino-terminusto carboxy-terminus e.g. in the following order: FR1, CDR1, FR2, CDR2,FR3, CDR3, FR4.

As used herein, the term “Complementarity Determining Regions” (CDRs;e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (e.g. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; and/or thoseresidues from a “hypervariable loop” (e.g. about residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain). Insome instances, a complementarity determining region can include aminoacids from both a CDR region defined according to Kabat and ahypervariable loop.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these maybe further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. A preferredclass of immunoglobulins for use in the present invention is IgG.

The heavy-chain constant domains that correspond to the differentclasses of antibodies are called [alpha], [delta], [epsilon], [gamma],and [mu], respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.As used herein antibodies are conventionally known antibodies andfunctional fragments thereof.

A “functional fragment” or “antigen-binding antibody fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hyper variable region(s) of anantibody, e.g., the CDR1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs. Preferably, the“antigen-binding region” comprises at least amino acid residues 4 to 103of the variable light (VL) chain and 5 to 109 of the variable heavy (VH)chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111of VH, and particularly preferred are the complete VL and VH chains(amino acid positions 1 to 109 of VL and 1 to 113 of VH; numberingaccording to WO 97/08320).

“Functional fragments”, “antigen-binding antibody fragments”, or“antibody fragments” of the invention include but are not limited toFab, Fab′, Fab′-SH, F(ab′)₂, and Fv fragments; diabodies; single domainantibodies (DAbs), linear antibodies; single-chain antibody molecules(scFv); and multispecific, such as bi- and tri-specific, antibodiesformed from antibody fragments. An antibody other than a“multi-specific” or “multi-functional” antibody is understood to haveeach of its binding sites identical. The F(ab′)₂ or Fab may beengineered to minimize or completely remove the intermolecular disulfideinteractions that occur between the CH1 and CL domains.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index.

Variants of the antibodies or antigen-binding antibody fragmentscontemplated in the invention are molecules in which the bindingactivity of the antibody or antigen-binding antibody fragment ismaintained.

“Binding proteins” contemplated in the invention are for exampleantibody mimetics, such as Affibodies, Adnectins, Anticalins, DARPins,Avimers, Nanobodies.

A “human” antibody or antigen-binding fragment thereof is hereby definedas one that is not chimeric (e.g., not “humanized”) and not from (eitherin whole or in part) a non-human species. A human antibody orantigen-binding fragment thereof can be derived from a human or can be asynthetic human antibody. A “synthetic human antibody” is defined hereinas an antibody having a sequence derived, in whole or in part, in silicofrom synthetic sequences that are based on the analysis of known humanantibody sequences. In silico design of a human antibody sequence orfragment thereof can be achieved, for example, by analyzing a databaseof human antibody or antibody fragment sequences and devising apolypeptide sequence utilizing the data obtained there from. Anotherexample of a human antibody or antigen-binding fragment thereof is onethat is encoded by a nucleic acid isolated from a library of antibodysequences of human origin (e.g., such library being based on antibodiestaken from a human natural source).

A “humanized antibody” or humanized antigen-binding fragment thereof isdefined herein as one that is (i) derived from a non-human source (e.g.,a transgenic mouse which bears a heterologous immune system), whichantibody is based on a human germline sequence; (ii) where amino acidsof the framework regions of a non-human antibody are partially exchangedto human amino acid sequences by genetic engineering or (iii)CDR-grafted, wherein the CDRs of the variable domain are from anon-human origin, while one or more frameworks of the variable domainare of human origin and the constant domain (if any) is of human origin.

A “chimeric antibody” or antigen-binding fragment thereof is definedherein as one, wherein the variable domains are derived from a non-humanorigin and some or all constant domains are derived from a human origin.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the term “monoclonal” indicatesthe character of the antibody as not being a mixture of discreteantibodies. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins. The term “monoclonal” is not to be construedas to require production of the antibody by any particular method. Theterm monoclonal antibody specifically includes chimeric, humanized andhuman antibodies.

An “isolated” antibody is one that has been identified and separatedfrom a component of the cell that expressed it. Contaminant componentsof the cell are materials that would interfere with diagnostic ortherapeutic uses of the antibody, and may include enzymes, hormones, andother proteinaceous or nonproteinaceous solutes.

As used herein, an antibody “binds specifically to”, is “specificto/for” or “specifically recognizes” an antigen of interest, e.g. atumor-associated polypeptide antigen target, is one that binds theantigen with sufficient affinity such that the antibody is useful as atherapeutic agent in targeting a cell or tissue expressing the antigen,and does not significantly cross-react with other proteins or does notsignificantly cross-react with proteins other than orthologs andvariants (e.g. mutant forms, splice variants, or proteolyticallytruncated forms) of the aforementioned antigen target. The term“specifically recognizes” or “binds specifically to” or is “specificto/for” a particular polypeptide or an epitope on a particularpolypeptide target as used herein can be exhibited, for example, by anantibody, or antigen-binding fragment thereof, having a monovalent K_(D)for the antigen of less than about 10⁻⁴ M, alternatively less than about10⁻⁵ M, alternatively less than about 10⁻⁶ M, alternatively less thanabout 10⁻⁷ M, alternatively less than about 10⁻⁸ M, alternatively lessthan about 10⁻⁹ M, alternatively less than about 10⁻¹⁰ M, alternativelyless than about 10⁻¹¹ M, alternatively less than about 10⁻¹² M, or less.An antibody “binds specifically to,” is “specific to/for” or“specifically recognizes” an antigen if such antibody is able todiscriminate between such antigen and one or more reference antigen(s).In its most general form, “specific binding”, “binds specifically to”,is “specific to/for” or “specifically recognizes” is referring to theability of the antibody to discriminate between the antigen of interestand an unrelated antigen, as determined, for example, in accordance withone of the following methods. Such methods comprise, but are not limitedto surface plasmon resonance (SPR), Western blots, ELISA-, RIA-, ECL-,IRMA-tests and peptide scans. For example, a standard ELISA assay can becarried out. The scoring may be carried out by standard colordevelopment (e.g. secondary antibody with horseradish peroxidase andtetramethyl benzidine with hydrogen peroxide). The reaction in certainwells is scored by the optical density, for example, at 450 nm. Typicalbackground (=negative reaction) may be 0.1 OD; typical positive reactionmay be 1 OD. This means the difference positive/negative is more than5-fold, 10-fold, 50-fold, and preferably more than 100-fold. Typically,determination of binding specificity is performed by using not a singlereference antigen, but a set of about three to five unrelated antigens,such as milk powder, BSA, transferrin or the like.

“Binding affinity” or “affinity” refers to the strength of the total sumof non-covalent interactions between a single binding site of a moleculeand its binding partner. Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a1:1 interaction between members of a binding pair (e.g. an antibody andan antigen). The dissociation constant “K_(D)” is commonly used todescribe the affinity between a molecule (such as an antibody) and itsbinding partner (such as an antigen) i.e. how tightly a ligand binds toa particular protein. Ligand-protein affinities are influenced bynon-covalent intermolecular interactions between the two molecules.Affinity can be measured by common methods known in the art, includingthose described herein. In one embodiment, the “K_(D)” or “K_(D) value”according to this invention is measured by using surface plasmonresonance assays using suitable devices including but not limited toBiacore instruments like Biacore T100, Biacore T200, Biacore 2000,Biacore 4000, a Biacore 3000 (GE Healthcare Biacore, Inc.), or a ProteOnXPR36 instrument (Bio-Rad Laboratories, Inc.).

The terms “nucleoside” and “nucleoside moiety” as use herein reference anucleic acid subunit including a sugar group and a heterocyclic base, aswell as analogs of such subunits, such as a modified or naturallyoccurring deoxyribonucleoside or ribonucleoside or any chemicalmodifications thereof. Other groups (e.g., protecting groups) can beattached to any component(s) of a nucleoside. Modifications of thenucleosides include, but are not limited to, 2′-, 3′- and 5′-positionsugar modifications, 5- and 6-position pyrimidine modifications, 2-, 6-and 8-position purine modifications, modifications at exocyclic amines,substitution of 5-bromo-uracil, and the like. Nucleosides can besuitably protected and derivatized to enable oligonucleotide synthesisby methods known in the field, such as solid phase automated synthesisusing nucleoside phosphoramidite monomers, H-phosphonate coupling orphosphate triester coupling.

A “nucleotide” or “nucleotide moiety” refers to a sub-unit of a nucleicacid which includes a phosphate group, a sugar group and a heterocyclicbase, as well as analogs of such subunits. Other groups (e.g.,protecting groups) can be attached to any component(s) of a nucleotide.The term “nucleotide”, may refer to a modified or naturally occurringdeoxyribonucleotide or ribonucleotide. Nucleotides in some cases includepurines and pyrimidines, which include thymidine, cytidine, guanosine,adenine and uridine. The term “nucleotide” is intended to include thosemoieties that contain not only the known purine and pyrimidine bases,e.g. adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U),but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, alkylated riboses or other heterocycles. Suchmodifications include, e.g., diaminopurine and its derivatives, inosineand its derivatives, alkylated purines or pyrimidines, acylated purinesor pyrimidines thiolated purines or pyrimidines, and the like, or theaddition of a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl,phenoxyacetyl, dimethylformamidine, dibutylformamidine,dimethylacetamidine, N,N-diphenyl carbamate, or the like. The purine orpyrimidine base may also be an analog of the foregoing; suitable analogswill be known to those skilled in the art and are described in thepertinent texts and literature. Common analogs include, but are notlimited to, 1-methyladenine, 2-methyladenine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine.

The term “oligonucleotide”, as used herein, refers to a polynucleotideformed from a plurality of linked nucleotide units as defined above. Thenucleotide units each include a nucleoside unit linked together via aphosphate linking group, or an analog thereof. The term oligonucleotidealso refers to a plurality of nucleotides that are linked together vialinkages other than phosphate linkages such as phosphorothioate linkagesor squaramide linkages. The oligonucleotide may be naturally occurringor non-naturally occurring. In some cases, the oligonucleotides mayinclude ribonucleotide monomers (i.e., may be oligoribonucleotides)and/or deoxyribonucleotide monomers.

The term “monosaccharide” as use herein refers to an open chained orcyclic compound of general formula C_(m)(H₂O)_(n) wherein m is 3, 4, 5,6, 7 or 8 and n is 2, 3, 4, 5 6, 7 or 8. However, the term alsoencompasses derivatives of these basic compounds wherein a OH group isreplaced by an NH₂ group (such as glucosamine), desoxysaccharides,wherein at least one OH group is replaced by H (e.g. desoxiribose).Preferred examples for monosaccharides are D-(+)-Glycerinaldehyd;D-(−)-Erythrose; D-(−)-Threose; D-(−)-Ribose; D-(−)-Arabinose;D-(+)-Xylose; D-(−)-Lyxose; D-(+)-Allose; D-(+)-Altrose; D-(+)-Glucose;D-(+)-Mannose; D-(−)-Gulose; D-(−)-Idose; D-(+)-Galactose; D-(+)-Talose;Dihydroxyaceton; D-Erythrulose; D-Ribulose; D-Xylulose; D-Psicose;D-Fructose; D-Sorbose; D-Tagatose. The term monosaccharide alsoencompasses monosaccharides which one, two, three or fourhydroxyl-groups are substituted.

The term “polysaccharides” refers to molecules comprising at least 2(two), preferably at least 5 (five), more preferably at least 10 (ten)monosaccharides which are connected via a glycosidic bond.

A carbohydrate as used herein encompasses a monosaccharide and apolysaccharide and derivatives thereof.

A polymer as used herein refers to macromolecules composed of manyrepeated organic subunits, however, which are no polysaccharides,oligonucleotides or peptides. Examples for polymers arePolyethylenglycole (PEG), polyoxyethylene (PEO) or polyglycerol (e.g.polyglycerol-polyricinoleate (PGPR).

The term “fluorophore” is well-known to the skilled person and refers tochemical compounds that re-emit light upon light excitation. Nonlimiting examples are CY₅, EDANS, Xanthene derivatives (e.g.fluorescein, Rhodamine, Oregon green, eosin, Texas red), Cyaninederivatives (e.g., indocarbocyanine, oxacarbocyanine, merocyanine),Squaraine derivatives (e.g., Seta, Se Tau, Square dyes), Naphthalenederivatives (e.g., dansyl or prodan derivatives), Coumarin derivatives,Oxadiazole derivatives, Anthracene derivatives (e.g., Anthraquinonessuch as DRAQ5, DRAQ7, CyTRAK Orange), Pyrene derivatives (e.g., cascadeblue), Oxazine derivatives (e.g., Nile red, Nile blue, Cresyl violet),Acridine derivatives (e.g., Proflavin, Acridine Orange, AcridineYellow), Arylmethine derivatives (e.g., Auramine, Crystal Violet,Malachite Green), or Tetrapyrrole derivatives (e.g., Parphin, Phthalocyanine, Bilirubin).

The term “aliphatic or aromatic residue” as used herein refers to analiphatic substituent, e.g. an alkyl residue which, however, can besubstituted by further aliphatic and/or aromatic substituents, e.g. analiphatic residue can be a nucleic acid, a peptide, a protein, anenzyme, a co-enzyme, an antibody, a nucleotide, an oligonucleotide, amonosaccharide, a polysaccharide, a polymer, a fluorophore, optionallysubstituted benzene, etc. as long as the direct link of such a moleculeto the core structure (in case of R₁, e.g., to the respective oxygen ofa compound of formula (III) or (V)) is aliphatic. An aromatic residue isa substitute, which direct link to the core structure is part of anaromatic system, e.g., an optionally substituted phenyl or pyridyl orpeptide, if the direct link of the peptide to the core structure is forexample via a phenyl-residue.

The term “antibody drug conjugate” or abbreviated ADC is well known to aperson skilled in the art, and, as used herein, refers to the linkage ofan antibody or an antigen binding fragment thereof with a drug, such asa chemotherapeutic agent, a toxin, an immunotherapeutic agent, animaging probe, and the like. As used herein, a “linker” is any chemicalmoiety that links an antibody or an antigen binding fragment thereofcovalently to the drug. As used herein, the term “linker drug conjugate”refers to a molecule or chemical group comprising or consisting of alinker as defined herein before, and a drug. In this regard, the term“linker drug conjugate” in general refers to that part of an antibodydrug conjugate which is not the antibody or an antigen binding fragmentthereof. In general, in a linker drug conjugate the linker is covalentlylinked to the drug.

Also described herein are “antibody fluorophore conjugates” orabbreviated AFC, which refers to the linkage of an antibody or anantigen binding fragment thereof with a fluorophore, such as, forexample, Cy5. The fluorophore may be linked to the antibody orantigen-binding fragment thereof through a linker, for example a linkeras described above in the context of an antibody drug conjugate. Theantibody fluorophore conjugate may comprise a “linker fluorophoreconjugate”. As used herein, the term “linker fluorophore conjugate”refers to a molecule or chemical group comprising or consisting of alinker as defined herein before, and a fluorophore. In this regard, theterm “linker fluorophore conjugate” in general refers to that part of anantibody drug conjugate which is not the antibody or an antigen bindingfragment thereof. In general, in a linker fluorophore conjugate thelinker is covalently linked to the fluorophore.

DETAILED DESCRIPTION

The invention provides a new chemoselective reaction of Cys residues in(unprotected) peptides, proteins, such as enzymes and co-enzymes (e.g.coenzyme A), antibodies or other thiol-comprising compounds with alkene-or alkyne-phosphonamidates. In one embodiment, the peptides, proteins,antibodies or other thiol-comprising compounds are unprotected. Inanother embodiment, the alkene- or alkyne-phosphonamidates are electrondeficient alkene- or alkyne-phosphonamidates. The resulting conjugateshave not been described in the literature previously.

Scheme 1, which is depicted in FIG. 36 , describes the general strategyfor a synthesis according to the present invention at the example ofethenyl or ethynyl phosphonites. R1 represents an optionally substitutedaliphatic or aromatic residue.

Scheme 2, which is depicted in FIG. 37 , shows the difference between aprocess known in the art (e.g., 15) and a process according to thepresent invention A) Sequential azide-azide couplings using alkynephosphonites; B) Staudinger-induced thiol-addition (the thiol additionmay be also denoted as “Michael addition”, as e.g. in FIG. 37B) for themodification of Cys residues according to the invention. Merely asexamples, ethenyl and ethynyl (diethyl)phosphonite were used.

It is submitted that the processes described herein allow to combine ahuge amount of different organic compounds in position R₁

and ●.

Furthermore, the invention refers to a method for bioconjugation of twocomplex molecules: a chemoselective reaction, which induces a secondchemoselective reaction for the conjugation to proteins. This concept isbased on the unique reactivity of an azide-building block with anunprotected alkyne or alkene phosphonite via the Staudinger-phosphonitereaction (SPhR) resulting in the generation of a, preferably,electron-deficient double or triple bond (Scheme 1 and 2B). Theresulting electrophilic system can subsequently be employed for thereaction with thiol-containing proteins and antibodies or furtherthiol-comprising compounds to deliver functional conjugates such asantibody or protein conjugates.

It is demonstrated with the attached results:

-   -   The synthesis of different alkene and alkyne phosphonites    -   (Chemoselective) Staudinger reactions with alkene and alkyne        phosphonites    -   Conjugation reactions of alkene- or alkyne-phosphonamidates with        thiol-containing molecules, including small molecules, peptides,        proteins and antibodies    -   Thiol addition to alkyne-phosphonamidates in aqueous systems        showed a high diastereoselectivity for the formation of the        Z-Product    -   Stability of these conjugates under physiologically relevant        conditions    -   Synthesis of conjugates comprising a cleavable group

This invention features several innovative aspects, which further easethe accessibility of conjugates such as antibody or protein conjugates,in particular with complex payloads and labels containing severalfunctional groups, with novel conjugation chemistry:

-   -   A new reaction for modifying thiols in small molecules,        polymers, proteins and antibodies, therefore    -   Unprecedented chemical structure at Cys-moiety    -   Two complex molecule (e.g. peptide and proteins or peptide and        antibody) can be connected by straightforward step-wise        chemoselective conjugations    -   No need of final protecting group manipulations after        installation of chemoselective handle (i.e., preferably        electron-deficient, alkene or alkyne-phosphonamidate) or after        the chemoselective conjugation    -   Linker with great variability (P-substituents can be varied,        various O-substituents at the phosphorus center, O-substituents        comprising a cleavable group)    -   High stability of conjugates as opposed to usual Maleimide        reagents; fast conjugation reactions    -   High stereoselectivity of the thiol addition to        alkyne-phosphonamidates

Generally, the process according to the present invention can be carriedout to conjugate different compounds such as small molecules (e.g.optionally substituted alkyl, phenyl or heterocycles), proteins,antibodies, oligonucleotides or polysaccharides with tags, proteinsoligonucleotides etc. To achieve this coupling, the present inventionrefers in a first aspect to a process for the preparation of conjugatesof formula (VII) comprising the steps of

-   -   (I) Reacting a compound of formula (III)

-   -   -   wherein        -   represents a double or triple bond;        -   X represents R₃—C when            is a triple bond        -   (thus, the structure is

-   -   -    or        -   X represents (R₃R₄)C when            is a double bond        -   (thus, the structure is

-   -   -   R₁ independently represents an optionally substituted            aliphatic or aromatic residue, such as phenyl; with            (C₁-C₈-alkoxy)_(n), wherein n is 1, 2, 3, 4, 5 or 6 with F,            with Cl, with Br, with I, with —NO₂, with —N(C₁-C₈-alkyl)H,            with —NH₂, with —N(C₁-C₃-alkyl)₂, with ═O, with            C₃-C₈-cycloalky, with optionally substituted phenyl            substituted C₁-C₈-alkyl such as

-   -   -    or optionally independently with C₁-C₈-alkyl,            (C₁-C₈-alkoxy)_(n), F, Cl, I, Br, —NO₂, —N(C₁-C₈-alkyl)H,            —NH₂, —N(C₁-C₈-alkyl)₂, substituted phenyl; or 5- or            6-membered heteroaromatic system such as pyridyl; preferably            C₁-C₈-alkyl, C₁-C₈-alkyl substituted with            (C₁-C₈-alkoxy)_(n), phenyl or phenyl substituted with —NO₂;        -   or            -   which may be again substituted at one of the                Nitrogen-ring-atoms with biotin or any other peptide,                protein, such as an enzyme or co-enzyme (e.g. coenzyme                A), antibody, protein tag, fluorophore, oligonucleotide,                or polysaccharide and wherein # represents the position                of O;        -   R₃ represents H or C₁-C₈-alkyl;        -   R₄ represents H or C₁-C₈-alkyl; and        -   with an azide of formula (IV)

-   -   -   wherein        -   ● represents an aliphatic or aromatic residue;        -   to prepare a compound of formula (V)

-   -   -   -   wherein ●,                , R₁, and X are as defined above.

    -   (II) Reacting a compound of formula (V) with a thiol-containing        molecule of formula (VI)

-   -   -   wherein            represents an optionally substituted C₁-C₈-alkyl, an            optionally substituted Phenyl, an optionally substituted            aromatic 5- or 6-membered heterocyclic system, an amino            acid, a peptide, a protein, an antibody, a saccharide, a            polysaccharide, a nucleotide, a oligonucleotide or a            polymer;        -   resulting in a compound of formula (VII)

-   -   -   wherein        -   represents a bond if            in a compound of formula (V) represents a double bond; or        -   represents a double bond if            in a compound of formula (V) represents a triple bond; and        -   , ●, R₁ and X are as defined above.

The invention also refers to a process comprising a step (a) prior tostep (1) of the process described above. Thus, such a process comprisesthe steps of

-   -   a) Reacting a compound of formula (I)

-   -   -   wherein R₁ and Hal are defined as above;        -   with an alpha unsaturated compound of formula (II)            comprising a double or triple bond in alpha-position

-   -   -   wherein        -   represents a double or triple bond;        -   X represents R₃—C when            is a triple bond; or        -   X represents (R₃R₄)C when            is a double bond;        -   R₃ represents H or C₁-C₈-alkyl; and        -   R₄ represents H or C₁-C₈-alkyl;        -   to form a compound of formula (III)

-   -   -   wherein        -   , X and R₁ are as defined above;        -   alternatively, reacting a compound of formula (I′)

-   -   -   wherein        -   R₅ independently represents C₁-C₈-alkyl;        -   Hal represents a halogen selected from the group consisting            of Cl, Br, I, preferably Cl;        -   with an alpha unsaturated compound of formula (II)            comprising a double or triple bond in alpha-position

-   -   -   wherein        -   represents a double or triple bond;        -   X represents R₃—C when            is a triple bond; or        -   X represents (R₃R₄)C when            is a double bond;        -   R₃ represents H or C₁-C₈-alkyl; and        -   R₄ represents H or C₁-C₈-alkyl;        -   to form a compound of formula (III′)

-   -   -   and reacting said compound of formula (III′) with R₁—OH        -   to form a compound of formula (III)

-   -   -   wherein        -   and X are defined as above and R₁ is as defined above but            not individually selected;

    -   (I) Reacting a compound of formula (III) with an azide of        formula (IV)

-   -   -   wherein        -   ● represents an aliphatic or aromatic residue;        -   to prepare a compound of formula (V)

-   -   -   wherein        -   ●,            , R₁ and X are as defined above;

    -   (II) Reacting a compound of formula (V) with a thiol-containing        molecule of formula (VI)

-   -   -   wherein            represents an optionally substituted C₁-C₈-alkyl, an            optionally substituted Phenyl, an optionally substituted            aromatic 5- or 6-membered heterocyclic system, an amino            acid, a peptide, a protein, an antibody, a saccharide, a            polysaccharide, a nucleotide, a oligonucleotide or a            polymer;        -   resulting in a compound of formula (VII)

-   -   -   wherein        -   represents a bond if            in a compound of formula (V) represents a double bond; or        -   represents a double bond if            in a compound of formula (V) represents a triple bond; and        -   , ●, R₁ and X are as defined above.

Preferably, in this process

represents a triple bond.

In one embodiment, the P-atom of compounds of formula (III), preferablywherein

represents a double bond, can be protected by BH₃ prior to theStaudinger reaction (e.g. for purification purposes) and can easily bedeprotected before the Staudinger reaction:

-   -   b) reacting a compound of formula (III)    -   to form a compound of formula (III)

-   -   wherein X and R₁ are as defined above;    -   with BH₃ to form a compound of formula (III′)

-   -   wherein X and R₁ are as defined above.

Deprotection of boran protected phosphonites of formula (III′) to formthe reactive P(III) species can be easily achieved by the addition of aweak base such as DABCO (1,4-Diazabicyclo[2.2.2]octan=Triethylendiamin(TEDA)).

Compounds of formula (III) can also be synthesized starting from PCI₃:

wherein R₁ is as defined herein.

The processes described herein can also be carried out with a compoundof formula (III*) instead of a compound (III)

Wherein V represents C₁-C₈-alkyl, preferably methyl, ethyl or propyl,more preferably methyl; and R₁, R₂ and R₃ are as defined for compound(III) above. For the preparation of compounds of formula (III*),compounds of formula (II*) can be used

wherein V, R₃ and R₄ are defined herein.

A process according to the invention with compound (III*) results incompounds of formula (V*)

wherein V represents C₁-C₈-alkyl, preferably methyl, ethyl or propyl,more preferably methyl; ● and R₁ are as defined for compound (V);

and compounds of formula (VII*)

wherein V represents C₁-C₈-alkyl, preferably methyl, ethyl or propyl,more preferably methyl; ●,

and R₁ are as defined for compound (VII). All steps for the processesdescribed herein for compounds of formula (V) and (VII) can be performedanalogously for compounds of formula (V*) and (VII*).

Accordingly, the present invention also relates to a process for thepreparation of alkene-phosphonamidates comprising the steps of:

(I) Reacting a compound of formula (III)

-   -   wherein    -   V represents C₁-C₈-alkyl, preferably methyl, ethyl or propyl,        more preferably methyl;    -   R₁ independently represents an optionally substituted aliphatic        or aromatic residue, such as phenyl; with (C₁-C₈-alkoxy)_(n),        wherein n is 1, 2, 3, 4, 5 or 6, with F, with Cl, with Br, with        I, with —NO₂, with —N(C₁-C₈-alkyl)H, with —NH₂, with        —N(C₁-C₈-alkyl)₂, with ═O, with C₃-C₈-cycloalkyl, with        optionally substituted phenyl substituted C₁-C₈-alkyl such as

or optionally independently with C₁-C₈-alkyl, (C₁-C₈-alkoxy)_(n), F, Cl,I, Br, —NO₂, —N(C₁-C₈-alkyl)H, —NH₂, —N(C₁-C₈-alkyl)₂, substitutedphenyl; or 5- or 6-membered heteroaromatic system such as pyridyl;preferably C₁-C₈-alkyl, C₁-C₈-alkyl substituted with (C₁-C₈-alkoxy)_(n),phenyl or phenyl substituted with —NO₂;

-   -   or        -   which may be again substituted at one of the            Nitrogen-ring-atoms with biotin or any other peptide,            protein, such as an enzyme or co-enzyme (e.g. coenzyme A),            antibody, protein tag, fluorophore, oligonucleotide, or            polysaccharide and wherein # represents the position of O;    -   R₃ represents H or C₁-C₈-alkyl;    -   R₄ represents H or C₁-C₈-alkyl; and    -   with an azide of formula (IV)

-   -   wherein    -   ● represents an aliphatic or aromatic residue;    -   to prepare a compound of formula (V*)

-   -   Wherein ●, V, and R₁ are as defined above;    -   X is (R₃R₄)C; and    -   R₃ and R₄ are as defined above;

(II) Reacting a compound of formula (V*) with a thiol-containingmolecule of formula (VI)

-   -   wherein        represents an optionally substituted C₁-C₈-alkyl, an optionally        substituted Phenyl, an optionally substituted aromatic 5- or        6-membered heterocyclic system, an amino acid, a peptide, a        protein, an antibody, a saccharide, a polysaccharide, a        nucleotide, a oligonucleotide or a polymer;    -   resulting in a compound of formula (VII*)

-   -   wherein    -   , ●, V, R₁ and X are as defined above.

One embodiment of the present invention also refers to compounds offormula (V*) and (VII*).

In the processes of the invention described herein it is not requiredthat the compound (V) or (V*) obtained in step (I) has exactly the samestructure as the compound (V) or (V*) used for reacting with thethiol-containing molecule of formula (VI) in step (II). In this respect,the ●, R₁ and/or X moieties of the compound (V) or (V*) may be modifiedbefore the compound (V) or (V*) is used for reacting with thethiol-containing molecule of formula (VI) in step (II). Suchmodification may be carried out as long as the ●, R₁ and/or X moietiesafter modification are still covered by the definitions disclosed hereinabove. As a merely illustrative example, as shown in the followingreaction scheme, the ● moiety of a compound A of formula (V) obtained instep (I) may be modified to give the compound B of formula (V), which isthen used for reacting with the thiol-containing molecule of formula(VI) in step (II):

wherein TFA⁻ is trifluoroacetate, Cy5 is the fluorescence dye Cy5, HATUis (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate), DIPEA is N,N-diisopropylethylamine and DMFis N,N-dimethylformamide.

In one preferred embodiment of a process according to the invention, R₁independently represents methyl, ethyl, propyl, butyl, phenyl,nitro-substituted phenyl, (C₁-C₂-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5or 6, more preferably 2-(2-methoxyethoxy)ethyl, phenyl, benzyl ornitro-substituted benzyl, methyl or ethyl, even more preferably methylor ethyl. In an even more preferred embodiment R₁ is the same.

In another preferred embodiment, R₁ can even be modified after thethiol-addition (step (II), for example, the substituent R₁ can comprisea triple bond as in

Which can be reacted with any desired organic compound-N₃ (such aspeptide-N₃, protein-N₃, such as an enzyme-N₃ or co-enzyme-N₃ (e.g.coenzyme A-N₃), antibody-N₃, protein tag-N₃, fluorophore-N₃,oligonucleotide-N₃, or polysaccharide-N₃ e.g. Biotin-N₃) to form atriazole-bridged complex, for example

Accordingly, R₁ may represent

wherein “compound” may represent a peptide, a protein, an enzyme, aco-enzyme (e.g. co-enzyme A), an antibody, a protein tag, a fluorophore,an oligonucleotide, a polysaccharide, or biotin; wherein # representsthe position of O.

In another preferred embodiment, R₁ is an optionally substitutedaliphatic or aromatic residue, such as phenyl; with (C₁-C₈-alkoxy)_(n),wherein n is 1, 2, 3, 4, 5 or 6 with F, with Cl, with Br, with I, with—NO₂, with —N(C₁-C₈-alkyl)H, with —NH₂, with —N(C₁-C₈-alkyl)₂, with ═O,with C₃-C₈-cycloalky, with optionally substituted phenyl substitutedC₁-C₈-alkyl such as

or optionally independently with C₁-C₈-alkyl, (C₁-C₈-alkoxy)_(n), F, Cl,I, Br, —NO₂, —N(C₁-C₈-alkyl)H, —NH₂, —N(C₁-C₈-alkyl)₂, substitutedphenyl; or 5- or 6-membered heteroaromatic system such as pyridyl;preferably C₁-C₈-alkyl, C₁-C₈-alkyl substituted with (C₁-C₈-alkoxy)_(n),phenyl or phenyl substituted with —NO₂.

Accordingly, R₁ may independently represent an optionally substitutedaliphatic or aromatic residue, such as phenyl. “Optionally substituted”in the “optionally substituted aliphatic or aromatic residue” refers tooptional substitution of the aliphatic or aromatic residue independentlywith any possible residue.

R₁ may represent C₁-C₈-alkyl optionally substituted with at least one of(C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, Br, I, —NO₂,—N(C₁-C₈-alkyl)H, —NH₂, —N(C₁-C₈-alkyl)₂, ═O, C₃-C₈-cycloalkyl,—S—S—(C₁-C₈-alkyl), hydroxy-(C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4,5 or 6, C₂-C₈-alkynyl or optionally substituted phenyl such as

wherein # represents the position of O in formula (III) or formula(III*).

R₁ may represent phenyl optionally independently substituted with atleast one of C₁-C₈-alkyl, (C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5or 6, F, Cl, I, Br, —NO₂, —N(C₁-C₈-alkyl)H, —NH₂ or —N(C₁-C₈-alkyl)₂.

R₁ may represent a 5- or 6-membered heteroaromatic system such aspyridyl.

R₁ may represent C₁-C₈-alkyl, C₁-C₈-alkyl substituted with—S—S—(C₁-C₈-alkyl), C₁-C₈-alkyl substituted with (C₁-C₈-alkoxy)_(n)wherein n is 1, 2, 3, 4, 5 or 6, C₁-C₈-alkyl substituted with optionallysubstituted phenyl, phenyl or phenyl substituted with —NO₂.

In some embodiments R₁ represents an aliphatic or aromatic residue whichis optionally substituted with —S—S—(C₁-C₈-alkyl). In a preferredembodiment, R₁ represents

wherein R₁₀ and R₁₁ independently represent hydrogen or C₁-C₈-alkyl; and# represents the position of O. In a more preferred embodiment R₁₀ andR₁₁ independently represent hydrogen, methyl or ethyl. In a still morepreferred embodiment, R₁ represents

wherein R₁₀ and R₁₁ independently represent hydrogen, methyl or ethyl;and # represents the position of O. In some of these embodiments R₁₀ andR₁₁ are both hydrogen. In some of these embodiments R₁₀ is hydrogen andR₁₁ is C₁-C₆-alkyl. In some of these embodiments R₁₀ is hydrogen and R₁₁is methyl or ethyl. Preferably, in these embodiments both R₁ are thesame.

In some embodiments R₁ represents C₁-C₈-alkyl substituted with phenyl,said phenyl being further substituted with

wherein Z is O or NH, and wherein # represents the position of saidphenyl. In some embodiments Z is O. In some embodiments Z is NH. TheC₁-C₈-alkyl in the

may be, for example, methyl, ethyl, propyl or butyl; preferably methyl,ethyl or propyl; more preferably methyl or ethyl; most preferablymethyl. In a preferred embodiment R₁ represents

wherein the C₁-C₈-alkyl may be, for example, methyl, ethyl, propyl orbutyl; preferably methyl, ethyl or propyl; more preferably methyl orethyl; most preferably methyl; wherein Z is O or NH, and wherein #represents the position of O. In another preferred embodiment R₁represents

wherein the C₁-C₈-alkyl may be, for example, methyl, ethyl, propyl orbutyl; preferably methyl, ethyl or propyl; more preferably methyl orethyl; most preferably methyl; wherein Z is O or NH, and wherein #represents the position of O. Preferably, in these embodiments both R₁are the same.

In some embodiments R₁ represents C₁-C₈-alkyl substituted with phenyl,said phenyl being further substituted with

and wherein # represents the position of said phenyl. In someembodiments R₁ represents C₁-C₈-alkyl substituted with phenyl, saidphenyl being further substituted with

wherein # represents the position of said phenyl. In a preferredembodiment R₁ represents

wherein # represents the position of O. In another preferred embodimentR₁ represents

wherein # represents the position of O. Preferably, in these embodimentsboth R₁ are the same.

In some embodiments R₁ represents an aliphatic or aromatic residue whichis optionally substituted with hydroxy-(C₁-C₈-alkoxy)_(n) wherein n is1, 2, 3, 4, 5 or 6. In a preferred embodiment R₁ is hydroxyethoxyethyl,more preferably —(CH₂)₂—O—(CH₂)₂—OH.

In some embodiments R₁ represents an aliphatic or aromatic residue whichis optionally substituted with C₂-C₈-alkynyl. In a preferred embodimentR₁ is homopropargyl.

In another preferred embodiment of a process according to the invention,

represents a double bond, X represents (R₃R₄)C, R₃ and R₄ independentlyrepresent H or C₁-C₈-alkyl and

represents a bond. In another preferred embodiment, R₃ and R₄ eachrepresent H.

In another preferred embodiment of a process according to the invention

represents a triple bond, X represents R₃—C, R₃ represents H orC₁-C₈-alkyl, more preferably H, and

represents a double bond.

In another preferred embodiment of a process according to the invention● represents an optionally substituted C₁-C₈-alkyl such as

r or

or an optionally substituted phenyl such as

wherein # represents the position of the —N₃ group of compounds offormula (IV), a radioactive or non-radioactive nuclide, biotin, anucleotide, an oligonucleotide, a polymer, a carbohydrate, an aminoacid, a peptide, an optionally substituted 5- or 6-memberedheteroaromatic system, a protein tag, or a fluorophore such as CY₅ orEDANS.

In another preferred embodiment of a process according to the invention● represents

-   -   a cyclic RGD peptide of structure (VIII) (c(RGDfK)

-   -   wherein    -   * represents the position of the N₃ group;    -   Biotin;    -   CY₅ or EDANS;    -   phenyl, optionally substituted with one, two, three, four or        five substituents independently selected from the group        consisting of C₁-C₈-alkyl, C₁-C₈-alkoxy, halogen, —CN, —NO₂,        —NH₂, —N(C₁-C₈-alkyl), —N(C₁-C₈-alkyl)₂-COOH, —COO(C₁-C₈-alkyl),        —O—C(O)—(C₁-C₈-alkyl), —C(O)N—(C₁-C₈-alkyl),        —N(H)—C(O)—(C₁-C₈-alkyl) preferably optionally substituted with        one substituent selected from the group consisting of        C₁-C₈-alkoxy, —COOH, —COO(C₁-C₈-alkyl and NO₂.    -   C₁-C₈-alkyl optionally substituted with at least one substituent        selected from the group consisting of C₃-C₈-cycloalkyl;        heterocyclyl with 3 to 8 ring members wherein the heteroatom(s)        are selected from N, O, S; C₁-C₈-alkoxy; halogen; —CN; —NO₂;        —NH₂; —N(C₁-C₈-alkyl); —N(C₁-C₈-alkyl)₂; —COOH;        —COO(C₁-C₈-alkyl); —O—C(O)—(C₁-C₈-alkyl); —CONH₂;        —C(O)N(C₁-C₈-alkyl)₂; —C(O)NH—(C₁-C₈-alkyl);        —N(H)—C(O)—(C₁-C₈-alkyl), preferably C₁-C₈-alkoxy, —COOH,        —COO(C₁-C₈-alkyl and NO₂, phenyl or a heteroaromatic system, a        monosaccharide, a polysaccharide, a peptide, a nucleotide, an        oligonucleotide, a polymer, an amino acid, a fluorophor, a        protein tag (substituent 1^(st) generation), wherein a        substituent 1^(st) generation may again optionally be        substituted with C₃-C₈-cycloalkyl; heterocyclyl with 3 to 8 ring        members wherein the heteroatom(s) are selected from N, O, S;        C₁-C₈-alkoxy; halogen; —CN; —NO₂; —NH₂; —N(C₁-C₈-alkyl);        —N(C₁-C₈-alkyl)₂; —COOH; —COO(C₁-C₈-alkyl);        —O—C(O)—(C₁-C₈-alkyl); —CONH₂; —C(O)N(C₁-C₈-alkyl)₂;        —C(O)NH—(C₁-C₈-alkyl); —N(H)—C(O)—(C₁-C₈-alkyl), preferably        C₁-C₈-alkoxy, —COOH, —COO(C₁-C₈-alkyl and NO₂, phenyl or a        heteroaromatic system (substituents 2^(nd) generation) and        wherein a substituent 2^(nd) generation may be substituted again        by at least one substituent selected from the same group and        wherein such substitution may go until generation 3, 4, 5, 6, 7,        8, 9 or 10.

In another preferred embodiment of a process according to the invention● represents

-   -   a cyclic RGD peptide of structure (VIII) (c(RGDfK)

-   -   wherein    -   * represents the position of the N₃ group;    -   Biotin;    -   CY₅ or EDANS;    -   phenyl, optionally substituted with one, two, three, four or        five substituents independently selected from the group        consisting of C₁-C₈-alkyl, C₁-C₈-alkoxy, halogen, —CN, —NO₂,        —NH₂, —N(C₁-C₈-alkyl), —N(C₁-C₈-alkyl)₂-COOH, —COO(C₁-C₈-alkyl),        —O—C(O)—(C₁-C₈-alkyl), —C(O)N—(C₁-C₈-alkyl),        —N(H)—C(O)—(C₁-C₈-alkyl) preferably optionally substituted with        one substituent selected from the group consisting of        C₁-C₈-alkoxy, —COOH, —COO(C₁-C₈-alkyl and NO₂.    -   C₁-C₈-alkyl optionally substituted with one, two, three, four or        five substituents independently selected from the group        consisting of phenyl which may be optionally substituted with        one, two, three, four or five substituents independently        selected from the group consisting of C₁-C₈-alkyl, C₁-C₈-alkoxy,        halogen, —CN, —NO₂, —NH₂, —N(C₁-C₈-alkyl),        —N(C₁-C₈-alkyl)₂-COOH, —COO(C₁-C₈-alkyl), —O—C(O)—(C₁-C₈-alkyl),        —C(O)N—(C₁-C₈-alkyl), —N(H)—C(O)—(C₁-C₈-alkyl), preferably        optionally substituted with one substituent selected from the        group consisting of C₁-C₈-alkoxy, —COOH, —COO(C₁-C₈-alkyl and        NO₂; C₁-C₈-alkoxy; halogen; —CN; —NO₂; —NH₂; —N(C₁-C₈-alkyl);        —N(C₁-C₈-alkyl)₂; —COOH; —COO(C₁-C₈-alkyl);        —O—C(O)—(C₁-C₈-alkyl); —C(O)N—(C₁-C₈-alkyl);        —N(H)—C(O)—(C₁-C₈-alkyl), preferably C₁-C₈-alkoxy, —COOH,        —COO(C₁-C₈-alkyl, —NO₂;

wherein # represents the N-position.

In another preferred embodiment of a process according to the invention● represents an optionally substituted phenyl such as

wherein # represents the position of the —N₃ group. TFA⁻ istrifluoroacetate.

In another preferred embodiment of a process according to the invention● represents an optionally substituted C₁-C₈-alkyl such as a linker, adrug, or a linker-drug conjugate.

In another preferred embodiment of a process according to the invention● represents an optionally substituted phenyl such as a linker, a drug,or a linker-drug conjugate.

In another preferred embodiment of a process according to the invention

represents an antibody, preferably a IgG-antibody, such as Cetuximab orTrastuzumab; a peptide, such as GFP protein, eGFP-protein, a tripeptide,e.g., a peptide of formula (IX)

-   -   Wherein # represents the position of S; or    -   optionally substituted C₁-C₈-alkyl such as

-   -   Wherein # marks the S-position.

In another preferred embodiment of a process according to the invention

represents

wherein # represents the position of S.

In an embodiment of a process according to the invention the

and the

are in the same molecule.

Accordingly, the present invention also relates to a process wherein acompound of formula (XX)

wherein the

and the

are in the same molecule as indicated by the arc connecting the ● andthe

,

is reacted with a compound of formula (III) as defined herein to give acompound of formula (VIIa):

-   -   wherein        represents a bond if        in a compound of formula (III) represents a double bond; or    -   represents a double bond if        in a compound of formula (III) represents a triple bond; and    -   , ●, R₁ and X are as defined herein.

Accordingly, the present invention also relates to a process wherein acompound of formula (XX)

wherein the

and the

are in the same molecule as indicated by the arc connecting the ● andthe

,

is reacted with a compound of formula (III*) as defined herein to give acompound of formula (VII*a):

-   -   wherein        , ●, V, R₁ and X are as defined herein.

In some embodiments the compound (XX) having the

and the

in the same molecule is a peptide, such as for example the BCL9 peptide.Accordingly, the compound of formula (VIIa) or (VII*a) obtained by theprocess may be a cyclic peptide, such as for example a cyclic peptidederived from the BCL9 peptide.

All steps for the processes described herein for compounds of formula(V), (V*), (VII) and (VII*) can be performed analogously for compoundsof formula (VIIa) and (VII*a).

The incorporation of both an azide and a thiol into the same moleculeprovides for an intramolecular Staudinger-induced thiol addition thatcan realize an intramolecular cyclization as exemplarily shown in theFIG. 38 .

Without wishing to be bound by any theory, it is assumed that first theazide is reacting with the electron-rich alkyne/alkene-phosphonite uponwhich the phosphonamidate is formed and an electron-pooralkyne/alkene-phosphonamidate is formed that undergoes a fastintramolecular thiol addition with the SH moiety.

One embodiment of the present invention also refers to compounds offormula (VIIa) and (VII*a).

Compounds

The invention also refers to compounds of formula (V)

Wherein R₁ and X and ● are as defined above.

The invention also refers to compounds of formula (V*)

-   -   wherein ●, V, R₁, and X are as defined above.

Preferably, in the compounds of formula (V) or (V*) ● represents anoptionally substituted C₁-C₈-alkyl such as

an optionally substituted phenyl such as

wherein # represents the N-position; a radioactive or non-radioactivenuclide, biotin, a nucleotide, an oligonucleotide, a polymer, acarbohydrate, an amino acid, a peptide, an optionally substitutedphenyl, an optionally substituted 5- or 6-membered heteroaromaticsystem, an optionally substituted C₁-C₈-alkyl, a protein tag or afluorophore such as CY₅.

Preferably, in the compounds of formula (V) or (V*) ● represents anoptionally substituted phenyl such as

wherein # represents the position of N. TFA⁻ is trifluoroacetate.

Preferably, in the compounds of formula (V) or (V*) ● represents anoptionally substituted C₁-C₈-alkyl such as a linker, a drug, or alinker-drug conjugate.

Preferably, in the compounds of formula (V) or (V*) ● represents anoptionally substituted phenyl such as a linker, a drug, or a linker-drugconjugate.

The invention also refers to compounds of formula (VII)

-   -   wherein    -   represents a bond and X represents (R₃R₄)C; or    -   represents a double bond and X represents R₃—C;    -   R₃ represents H or C₁-C₈-alkyl;    -   R₄ represents H or C₁-C₈-alkyl;    -   represents an optionally substituted C₁-C₈-alkyl, an optionally        substituted Phenyl, an optionally substituted aromatic 5- or        6-membered heterocyclic system, an amino acid, a peptide, a        protein, an antibody, a saccharide, a polysaccharide, a        nucleotide, a oligonucleotide or a polymer;        -   ● represents an aliphatic or aromatic residue;        -   R₁ independently represents an optionally substituted            aliphatic or aromatic residue, such as phenyl; with            (C₁-C₈-alkoxy)_(n), wherein n is 1, 2, 3, 4, 5 or 6 with F,            with Cl, with Br, with I, with —NO₂, with —N(C₁-C₈-alkyl)H,            with —NH₂, with —N(C₁-C₈-alkyl)₂, with ═O, with            C₃-C₈-cycloalky, with optionally substituted phenyl            substituted C₁-C₈-alkyl such as

or optionally independently with C₁-C₈-alkyl, (C₁-C₈-alkoxy)_(n), F, Cl,I, Br, —NO₂, —N(C₁-C₈-alkyl)H, —NH₂, —N(C₁-C₈-alkyl)₂, substitutedphenyl; or 5- or 6-membered heteroaromatic system such as pyridyl;preferably C₁-C₈-alkyl, C₁-C₈-alkyl substituted with (C₁-C₈-alkoxy)_(n),phenyl or phenyl substituted with —NO₂;

The invention also refers to compounds of formula (VII*)

-   -   wherein    -   represents an optionally substituted C₁-C₈-alkyl, an optionally        substituted Phenyl, an optionally substituted aromatic 5- or        6-membered heterocyclic system, an amino acid, a peptide, a        protein, an antibody, a saccharide, a polysaccharide, a        nucleotide, a oligonucleotide or a polymer;        -   ● represents an aliphatic or aromatic residue;        -   R₁ independently represents an optionally substituted            aliphatic or aromatic residue, such as phenyl; with            (C₁-C₈-alkoxy)_(n), wherein n is 1, 2, 3, 4, 5 or 6 with F,            with Cl, with Br, with I, with —NO₂, with —N(C₁-C₈-alkyl)H,            with —NH₂, with —N(C₁-C₈-alkyl)₂, with ═O, with            C₃-C₈-cycloalky, with optionally substituted phenyl            substituted C₁-C₈-alkyl such as

-   -   -    or optionally independently with C₁-C₈-alkyl,            (C₁-C₈-alkoxy)_(n), F, Cl, I, Br, —NO₂, —N(C₁-C₈-alkyl)H,            —NH₂, —N(C₁-C₈-alkyl)₂, substituted phenyl; or 5- or            6-membered heteroaromatic system such as pyridyl; preferably            C₁-C₈-alkyl, C₁-C₈-alkyl substituted with            (C₁-C₈-alkoxy)_(n), phenyl or phenyl substituted with —NO₂;        -   V represents C₁-C₈-alkyl, preferably methyl, ethyl or            propyl, more preferably methyl;        -   X represents (R₃R₄)C        -   R₃ represents H or C₁-C₈-alkyl; and        -   R₄ represents H or C₁-C₈-alkyl.

Preferably, in the compounds of formula (VII) or (VII*) ● represents anoptionally substituted C₁-C₈-alkyl such as

an optionally substituted phenyl such as

a radioactive or non-radioactive nuclide, biotin, a nucleotide, anoligonucleotide, a polymer, a carbohydrate, an amino acid, a peptide, anoptionally substituted phenyl, an optionally substituted 5- or6-membered heteroaromatic system, an optionally substituted C₁-C₈-alkyl,a protein tag or a fluorophore such as CY₅ or EDANS.

Preferably, in the compounds of formula (VII) or (VII*) ● represents anoptionally substituted phenyl such as

wherein # represents the position of N. TFA⁻ is trifluoroacetate.

Preferably, in the compounds of formula (VII) or (VII*) ● represents anoptionally substituted C₁-C₈-alkyl such as a linker, a drug, or alinker-drug conjugate.

Preferably, in the compounds of formula (VII) or (VII*) ● represents anoptionally substituted phenyl such as a linker, a drug, or a linker-drugconjugate.

Preferably, in the compounds of formula (VII) or (VII*)

represents an antibody, preferably a IgG-antibody, more preferably aCetuximab or a Trastuzumab; a peptide, preferably GFP protein oreGFP-protein or a tripeptide, more preferably a peptide of formula (IX)or C₁-C₈-alkyl.

Preferably, in the compounds of formula (VII) or (VII*)

represents

wherein # represents the position of S.

Preferred conjugates of formula (VII) or formula (VII*) are conjugateswherein

-   -   represents an antibody and    -   ● represents a protein tag or a fluorophore such as CY₅ or        EDANS, or a protein.

Further preferred conjugates of formula (VII) are conjugates wherein

-   -   represents a protein and    -   ● represents a protein tag or a fluorophore such as CY₅ or        EDANS, an antibody or a protein.

One preferred embodiment are conjugates of formula (VII) wherein

-   -   represents a protein and    -   ● represents a protein.

Further, preferred conjugates of formula (VII) or formula (VII*) areconjugates wherein

-   -   represents an antibody and    -   ● represents a linker, a drug, or a linker-drug conjugate.

The invention also refers to compounds of formula (VIIa)

wherein ● and

are in the same molecule as indicated by the arc connecting the ● andthe

, and wherein ●,

,

, X and R₁ are as defined herein, in particular as defined with regardto compound (VII). Preferably, the compound (VIIa) is a cyclic peptide,such as for example a cyclic peptide derived from the BCL9 peptide.

The invention also refers to compounds of formula (VII*a)

wherein ● and

are in the same molecule as indicated by the arc connecting the ● andthe

, and wherein ●,

, V, X and R₁ are as defined herein, in particular as defined withregard to compound (VII*). Preferably, the compound (VII*a) is a cyclicpeptide, such as for example a cyclic peptide derived from the BCL9peptide.

The following compounds of formula (VII) are also preferred:

the compound depicted in FIG. 30 ;

A fluorescently labeled ASGP-R addressing Cys conjugate of formula (X)which can be produced via the modular addition to vinyl phosphonamidatesis depicted in FIG. 39 .

The following compounds of formula (VII) are also preferred:

wherein LD represents a linker drug conjugate having the structure

and # represents the position of the N; and

Moreover, also compounds provided herein as examples in the examplesection for compounds of formula (VII) are preferred.

The skilled person understands that embodiments according to theinvention can be combined with each other with the proviso that acombination which would contravene any natural law is excluded.

Synthesis of Phosphonamidate of Formula (V)

Step a)

General procedure for the preparation of alkenyl or alkynylphosphonamidates by Staudinger phosphonite reaction requires thereaction of an alkenyl- or alkynylmagnesiumbromide of formula (II) witha dialkyl halogenchlorophosphite of formula (I), preferably achlorophosphonite, below −20° C., e.g. between −100° C. and −40° C.,preferably between −90° C. and −50° C. (e.g. around 87° C.). Preferably,the reaction is carried out under inert gas such as argon. “Inert” inthis situation refers to a gas which will not react with any of theeducts or products of this reaction under the given reaction conditions.Of course, the reaction time depends on the reaction volume and amountof substance. However, as a guideline, the reaction time should be in arange from 2 min to 4 h. The amounts of compound of formula (I) and (II)should be in a range from 5:1 to 1:5 such as 2:1 to 1:2, e.g., around1:1.

Step (I)

The reaction of a compound of formula (III) with an azide of formula(IV) can be performed at room temperature, i.e. around 25° C. However,the reaction can also be carried out at temperatures in a range from 0°C. to 50° C. The reaction time depends on the reaction volume and theamount of substance. However, as a guideline, the reaction should becarried out in a time frame from 1 h to 72 h. The amounts of compound offormula (III) and (IV) should be in a range from 5:1 to 1:5 such as 2:1to 1:2, e.g., around 1:1.

Preferred solvents for step (1) described herein is carried out in apolar aprotic solvent system such as tetrahydrofurane (THF),dimethylformamide (DMF), acetonitrile (MeCN), acetone, dimethylsulfoxide (DMSO), ethyl acetate (EtOAc), N-methylpyrrolidone or mixturesthereof, preferably THF, DMF, MeCN, THF/DMF, THF/MeCN; or a mixture of apolar unprotic solvent and a non-polar solvent such as hexane, toluene,benzene, 1,4-dioxane, chloroform, diethylether or dichloromethane (DCM),preferably THF/toluene. Step (1) may be also carried out in an aqueousmedium, for example in water or in an aqueous buffer, such as forexample phosphate-buffered saline (PBS),tris(hydroxymethyl)-aminomethane (TRIS) or bicarbonate.

Procedure for Base Mediated Hydrothiolations of Electron-DeficientPhosphonamidate Alkynes

Step (II)

Phosphonamidate of formula (V) and a base (and additive where required)can be suspended in a respective solvent. Then a thiol of formula (VI)can be added, e.g., via a microliter syringe and the mixture is allowedto react at room temperature, i.e. around 25° C. However, the reactioncan also be carried out at temperatures in a range from 0° C. to 50° C.The reaction time depends on the reaction volume and the amount ofsubstance. However, as a guideline, the reaction should be carried outin a time frame from 0.1 h (hours) to 10 h, e.g., in a time frame from0.1 h to 3 h or even within a time frame between 0.1 h and 1 h.

In a preferred embodiment, step (II) described herein is carried out inthe presence of a weak base. Preferred weak bases are carbonates such asammonium (NH₄)₂CO₃, Na₂CO₃, Rb₂CO₃, K₂CO₃, or Cs₂CO₃ or correlatinghydrogencarbonates thereof (e.g. NaHCO₃ etc.); and weak Nitrogencontaining bases such as triethylamine Et₃N (pK_(a) 10.76 at 25° C.).Preferably, a base with a pK_(a) value within the range of 7.5 to 11.5is used.

The solvent (system) can be chosen from a wide range of solvents. Thesolvent can be a polar aprotic solvent system such as tetrahydrofurane(THF), dimethylformamide (DMF), acetonitrile (MeCN), acetone, dimethylsulfoxide (DMSO), ethyl acetate (EtOAc), N-methylpyrrolidone or mixturesthereof, preferably THF, DMF, DMSO; non-polar solvents such as hexane,toluene, benzene, 1,4-dioxane, chloroform, diethylether ordichloromethane (DCM), preferably DCM; polar protic solvents such aswater, ethanol, isopropanol, methanol, n-butanol, preferably ethanol; ormixtures thereof, e.g., DMF/water. The solvent may be also an aqueousmedium, such as for example water or an aqueous buffer, such as forexample phosphate-buffered saline (PBS),tris(hydroxymethyl)-aminomethane (TRIS) or bicarbonate.

EXAMPLES General Procedure for the Preparation of AlkynylPhosphonamidates

In a flame dried Schlenk flask under an atmosphere of argon a solutionof ethynyl magnesiumbromide (0.5 M in THF, 2 mL, 1 mmol) was cooled to−78° C. in a bath of dry ice/acetone. The diethyl chlorophosphite (157mg, 144 μL, 1 mmol) was added dropwise via a syringe. The solution wasstirred at −78° C. for 30 minutes, then warmed up to room temperatureand subsequently stirred for another 1.5 hours. Afterwards 3 mL of dryTHF and azide (1 mmol) was added and the solution was stirred at roomtemperature for 24 hours. Then H₂O (5 mL) was added and the solution wasstirred for another 24 hours open to air. After removal of the solventunder reduced pressure the crude mixture was analyzed by ³¹P NMR.

Synthesis of Vinyl Phosphonites General Procedure A for the Synthesis ofVinyl Phosphonites from Phosphorous Trichloride

A flame-dried Schlenkflask was charged with 1.50 mmol (1.0 eq.) ofphosphorous trichloride in 20 ml of dry toluene and cooled to −78° C.3.3 mmol of pyridine (2.2 eq.) and a solution of 3.3 mmol of the alcohol(2.2 eq.) in 5 ml Et₂O were added drop wise. The resulting suspensionwas allowed to warm to room temperature, stirred for another 30 min andcooled again to −78° C. 1.65 mmol (1.1 eq.) of Vinylgrignard (1.0 M inTHF) was added and the reaction was stirred at room temperature for twohours. Finally 2.25 mmol (1.5 eq.) of borane (1.0 M in THF) were addedat 0° C. and stirred for another hour. The crude product was dry packedon a silica column for purification.

General Procedure B for the Synthesis of Vinyl Phosphonites fromBis(Diisopropylamino)Chlorophosphine

A flame-dried Schlenkflask was charged with 1.5 mmol (1.0 eq.)Bis(diisopropylamino)chlorophosphine, dissolved in 200 μl of dry THF andcooled to −78° C. 1.65 mmol (1.1 eq.) of Vinylgrignard (1.0 M in THF)was added and the reaction was stirred at room temperature for 30minutes. A solution of 3.3 mmol vacuum dried alcohol (2.2 eq.) in 1 mlof dry THF or MeCN and 3.3 mmol (2.2 eq.) Tetrazole (0.45 M in MeCN) wasadded. The resulting suspension was stirred at room temperatureovernight. Finally 2.25 mmol (1.5 eq.) of borane (1.0 M in THF) wereadded at 0° C. and stirred for another hour. The crude product was drypacked on a silica column for purification.

General Procedure C for the Synthesis of Vinyl Phosphonamidates fromDiethylchloro Phosphite, Vinyl Grignard Reagent and Different Azides

A 25-ml Schlenk flask was charged with 1.71 ml vinylmagnesium bromide(0.7 M in THF, 1.20 mmol, 1.2 eq.) under an argon atmosphere, cooled to−78° C. and 140 μl diethyl chlorophosphite (1.00 mmol, 1.0 eq.) wereadded drop wise. The yellowish solution was allowed to warm to 0° C.,stirred for another two hours and 1.00 mmol of azide (1.0 eq.) dissolvedin 3.2 ml of THF was added and stirred over night at room temperature. 5ml of water were added and stirred for another 24 h. The solvents wereremoved under reduced pressure and the crude product was purified byflash column chromatography on silica gel.

Ethyl-N-phenyl-P-ethynyl-phosphonamidate

Ethyl N-phenyl-P-ethynyl-phosphonamidate was prepared after “generalprocedure for the preparation of alkenyl or alkynyl phosphonamidates” on5 mmol scale from phenyl azide (595 mg, 5 mmol). The crude mixture waspurified by silica gel column chromatography eluting with hexane/ethylacetate. The product was obtained as colourless solid in a yield of 430mg (2.1 mmol, 42%). ¹H NMR (300 MHz, Chloroform-d): 5=7.28 (t, J=7.7 Hz,2H, ArH), 7.11 (d, J=8.0 Hz, 2H, ArH), 7.02 (t, J=7.3 Hz, 1H, ArH), 6.74(d, J=7.6 Hz, 1H, NH), 4.55-3.93 (m, 2H, CH₂), 2.89 (d, J=12.8 Hz, 1H,CH), 1.39 (t, J=7.0 Hz, 3H, CH₃) ppm. ¹³C NMR (75 MHz, Chloroform-d):5=138.99, 129.39, 122.42, 118.23, 118.13, 88.10, 87.45, 76.34 (d,J=273.3 Hz), 62.26 (d, J=5.2 Hz), 16.23 (d, J=7.4 Hz) ppm. ³¹P NMR (122MHz, Chloroform-d): δ=−9.17 ppm. HRMS ESI-TOF m/z [M+H]⁺=210.0678(calcd.); 210.0687 (found).

Ethyl-N-benzyl-P-ethynyl-phosphonamidate

Ethyl N-benzyl-P-ethynyl-phosphonamidate was prepared after “generalprocedure for the preparation of alkenyl or alkynyl phosphonamidates”from benzyl azide (133 mg, 125 μL, 1 mmol). The crude mixture waspurified by silica gel column chromatography eluting with hexane/ethylacetate. The product was obtained as colourless solid in a yield of 37mg (0.17 mmol, 17%). ¹H NMR (300 MHz, Chloroform-d): δ=7.51-7.18 (m, 5H,ArH), 4.26-4.04 (m, 4H, 2×CH₂), 3.34 (s, 1H, CH), 2.91 (d, J=12.7 Hz,1H, NH), 1.34 (t, J=7.1 Hz, 3H, CH₃) ppm. ¹³C NMR (75 MHz,Chloroform-d): δ=138.99, 138.90, 128.71, 127.62, 127.54, 87.77, 87.16,76.83 (d, J=260.0 Hz), 62.03 (d, J=5.1 Hz), 44.86, 16.25 (d, J=7.3 Hz)ppm. ³¹P NMR (122 MHz, Chloroform-d): δ=−2.76 ppm. HRMS ESI-TOF m/z[M+H]⁺=224.0835 (calcd.); 224.0835 (found).

Ethyl-N-phenyl-P-vinyl-phosphonamidate

The compound was synthesized according to the general procedure C from1.15 ml diethyl chlorophosphite (8 mmol). The pure phosphonamidate waspurified by flash column chromatography (EtOAc) and obtained as a whitesolid. (675 mg, 3.20 mmol, 40.0%) ¹H NMR (600 MHz, Chloroform-d) δ=7.24(dd, J=8.5, 7.3, 2H), 7.05-7.01 (m, 2H), 6.99 (d, J=5.8, 1H), 6.94 (tt,J=7.3, 1.1, 1H), 6.33-6.23 (m, 2H), 6.10 (ddd, J=50.3, 9.6, 5.1, 1H),4.29-4.04 (m, 2H), 1.35 (t, J=7.1, 3H). ¹³C NMR (151 MHz, Chloroform-d)δ=140.43, 134.44, 129.28, 127.51 (d, J=172.7), 121.26, 117.31 (d,J=6.6), 60.44 (d, J=6.2), 16.22 (d, J=7.0). ³¹P NMR (122 MHz,Chloroform-d) δ=15.68. HRMS for C₁₀H₁₅NO₂P⁺ [M+H]⁺ calcd: 212.0835,found: 212.0839.

Ethyl-N-(4-carboxy-phenyl)-P-vinyl-phosphonamidate

The compound was synthesized according to the general procedure C from288 μl diethyl chlorophosphite (2 mmol). The pure phosphonamidate waspurified by flash column chromatography (CH₂Cl₂/MeOH, 9:1 to 4:1) andobtained as a white solid. (173 mg, 0.68 mmol, 34.0%)

¹H NMR (600 MHz, DMSO-d₆) δ=8.37 (d, J=7.9, 1H), 7.80 (d, J=8.7, 2H),7.12 (d, J=8.7, 2H), 6.42-6.04 (m, 3H), 4.11-3.94 (m, 2H), 1.26 (t,J=7.0, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ=167.56, 146.36, 135.00, 131.21,129.04 (d, J=165.8), 123.06, 117.00 (d, J=6.9), 60.84 (d, J=5.7), 16.61(d, J=6.3). ³¹P NMR (122 MHz, DMSO-d₆) δ=14.36. HRMS for C₁₁H₁₅NO₄P⁺[M+H]⁺ calcd: 256.0733, found: 256.0723.

Ethyl-N-benzyl-P-vinyl-phosphonamidate

The compound was synthesized according to the general procedure C from290 μl diethyl chlorophosphite (2 mmol). The pure phosphonamidate waspurified by flash column chromatography (EtOAc) and obtained as acolourless oil. (155 mg, 0.69 mmol, 34.3%)

¹H NMR (300 MHz, Chloroform-d) δ=7.36-7.21 (m, 5H), 6.33-5.88 (m, 3H),4.16-3.90 (m, 4H), 3.21 (d, J=8.5, 1H), 1.28 (t, J=7.1, 3H). ¹³C NMR (75MHz, Chloroform-d) δ=139.65 (d, J=5.9), 133.21 (d, J=1.5), 129.45,128.46, 127.20, 127.17, 60.11 (d, J=5.7), 44.58, 16.27 (d, J=6.7). ³¹PNMR (122 MHz, Chloroform-d) δ=20.52. HRMS for C₁₁H₁₇NO₂P⁺ calcd:226.0991, found: 226.1003

Ethyl-N-(2-nitro-Benzyl)-P-vinyl-phosphonamidate

The compound was synthesized according to the general procedure C from120 μl diethyl chlorophosphite (0.83 mmol). The pure phosphonamidate waspurified by flash column chromatography (2% MeOH in CH₂Cl₂) and obtainedas a brown oil. (125 mg, 0.46 mmol, 55.4%)

¹H NMR (300 MHz, Chloroform-d) δ=8.03 (d, J=8.1, 1H), 7.73-7.57 (m, 2H),7.45 (t, J=7.6, 1H), 6.31-5.83 (m, 3H), 4.39 (dd, J=11.2, 7.7, 2H),4.12-3.85 (m, 2H), 3.65 (q, J=8.6, 1H), 1.26 (t, J=7.1, 3H). ¹³C NMR (75MHz, Chloroform-d) δ=148.09, 135.45 (d, J=4.2), 133.83, 133.52 (d,J=1.6), 131.10, 128.41, 128.26 (d, J=169.7), 124.95, 60.35 (d, J=5.7),42.42 (d, J=1.3), 16.22 (d, J=6.7). ³¹P NMR (122 MHz, Chloroform-d)δ=20.63. HRMS for C₁₁H₁₆N₂O₄P⁺ calcd: 271.0842, found: 271.0851.

Ethyl-N-(3-phenyl-propyl)-P-vinyl-phosphonamidate

The compound was synthesized according to the general procedure C from290 μl diethyl chlorophosphite (2 mmol). The pure phosphonamidate waspurified by flash column chromatography (EtOAc) and obtained as acolourless oil. (165 mg, 0.65 mmol, 32.5%)

¹H NMR (300 MHz, Chloroform-d) δ=7.28 (dd, J=8.1, 6.2, 2H), 7.23-7.11(m, 3H), 6.28-5.89 (m, 3H), 4.04 (qt, J=10.2, 7.2, 2H), 2.92 (dq, J=9.1,7.0, 2H), 2.84-2.70 (m, 1H), 2.70-2.60 (m, 2H), 1.82 (p, J=7.3, 2H),1.31 (t, J=7.1, 3H). ¹³C NMR (75 MHz, Chloroform-d) 5=141.28, 132.98 (d,J=1.5), 128.42 (d, J=169.0), 128.34, 128.24, 125.88, 59.95 (d, J=5.7),40.23, 33.53 (d, J=5.6), 32.86, 16.32 (d, J=6.7). ³¹P NMR (122 MHz,Chloroform-d) δ=20.82. HRMS for C₁₁H₁₆N₂O₄P⁺ calcd: 271.0842, found:271.0851. HRMS for C₁₃H₂₁NO₂P⁺ calcd: 254.1304, found: 254.1312.

Ethyl-N-cyclohexyl-P-vinyl-phosphonamidate

The compound was synthesized according to the general procedure C from140 μl diethyl chlorophosphite (1 mmol). The pure phosphonamidate waspurified by flash column chromatography (1.5% MeOH in CH₂Cl₂) andobtained as a colourless oil. (70 mg, 0.32 mmol, 32.2%)

¹H NMR (600 MHz, Chloroform-d) δ=6.25-5.93 (m, 3H), 4.14-3.97 (m, 2H),2.96 (dqd, J=13.8, 9.6, 8.1, 4.2, 1H), 2.51 (t, J=9.6, 2H), 1.97-1.84(m, 2H), 1.74-1.65 (m, 1H), 1.57 (dt, J=13.0, 3.9, 1H), 1.32 (t, J=7.1,3H), 1.30-1.09 (m, 5H). ¹³C NMR (75 MHz, Chloroform-d) 5=132.56 (d,J=1.8), 129.30 (d, J=168.8), 59.80 (d, J=5.9), 49.71, 36.03, 25.24,24.96, 16.32 (d, J=6.8). ³¹P NMR (122 MHz, Chloroform-d) δ=19.34. HRMSfor C₁₀H₂₁NO₂P⁺ calcd: 218.1304, found: 218.1302.

Staudinger-Induced Thiol-Additions with Alkynyl-Phosphonites Synthesisof Diethyl-Alkynyl-Phosphonite and Reaction with Different Azides

(Step b)

Diethyl-alkynyl-phosphonite was synthesized according to publishedprotocols (13) and reacted with different aliphatic and aromatic azides(Scheme 3). The formation of the desired alkynyl-phosphonamidates wasmonitored by ³¹P-NMR (see Table 1 for conversions for different azidesubstrates).

TABLE 1 Substrate scope for the Staudinger phosphonite reaction ofdiethyl-alkynyl- phosphonite (values in %) n.d. = not detected);determined by ³¹P-NMR. Entry R =

1

94 n. d. n. d. 2

91 n. d. n. d. 3

70 n. d. 25 4

44 n. d. n. d. 5

70  4 25 6

73 10 14 7

76  5 18 8

45 17 28

N-phenyl- and N-benzyl-phosphonamidates were isolated by columnchromatography in yields of 41% and 17% respectively. The highestconversions were obtained in THF (Table 2).

TABLE 2 Influence of the solvent on the Staudinger phosphonite reactionbetween diethyl-alkynyl-phosphonite and phenylazide. Entry SolventConversion 1 THF 94 2 THF/DMF 86 3 THF/Acetonitrile 91 4 THF/Toluene 88

General Procedure for Base Mediated Hydrothiolations of PhosphonamidateAlkynes or Alkenes

To a capped vial Ethyl N-phenyl-P-ethynyl-phosphonamidate (10 mg, 0.05mmol) and the respective base (and additive where required) was added.The mixture was suspended in 200 μL of respective solvent. Thenethanethiol (3.1 mg, 3.6 μL, 0.05 mmol) was added via a microlitersyringe and the mixture was stirred at room temperature for 3 hours.Afterwards the mixture was diluted with CH₂Cl₂ (5 mL) and H₂O (5 mL) wasadded. After extraction the phases were separated and the aqeuous layerwas extracted three times with CH₂Cl₂ (5 mL). The combined organiclayers were washed two times with H₂O (5 mL) and with brine (5 mL).After removal of the solvent the crude mixture was analyzed by ¹H NMRand ³¹P NMR. The preparation of alkene phosphonamidates is analogous tothe preparation of alkyne phosphonamidates.

Ethyl-N-phenyl-P-(2-ethylsulfanyl)-ethenyl-phosphonamidate

To a capped vial Ethyl N-phenyl-P-ethynyl-phosphonamidate (10 mg, 0.05mmol) and potassium carbonate (2.8 mg, 0.02 mmol) was added. The mixturewas suspended in a 1 to 1 mixture of DMF/H₂O (200 μL). Then ethanethiol(3.1 mg, 3.6 μL, 0.05 mmol) was added via a microliter syringe and themixture was stirred at room temperature for 3 hours. Afterwards themixture was diluted with CH₂Cl₂ (5 mL) and H₂O (5 mL) was added. Afterextraction the phases were separated and the aqeuous layer was extractedthree times with CH₂Cl₂ (5 mL). The combined organic layers were washedtwo times with H₂O (5 mL) and with brine (5 mL). After removal of thesolvent under reduced pressure the product was obtained in a yield of 12mg (0.044 mmol, 89%). ¹H NMR (300 MHz, Chloroform-d): δ=7.45 (dd,J=21.7, 16.7 Hz, 1H, P—CH, E), 7.30-7.17 (m, 3H, ArH), 7.06 (d, J=12.5Hz, 1H, S—CH, Z), 7.01-6.90 (m, 2H, ArH), 5.75 (dd, J=16.7, 12.5 Hz, 1H,P—CH, Z), 4.35-4.00 (m, 2H, OCH₂), 2.75 (q, J=7.5 Hz, 2H, SCH₂), 1.36(t, J=7.0 Hz, 3H, OCH₂CH₃), 1.28 (t, J=7.5 Hz, 3H, SCH₂CH₃) ppm. ¹³C NMR(75 MHz, Chloroform-d): δ=150.40, 140.11, 129.31, 121.50, 117.47 (d,J=6.4 Hz), 60.61 (d, J=6.0 Hz), 29.59, 25.90, 16.42 (d, J=6.9 Hz),15.53, 13.75 ppm. ³¹P NMR (122 MHz, Chloroform-d) δ 15.13, 14.35 ppm.HRMS ESI-TOF m/z [M+H]⁺=272.0869 (calcd.); 272.0855 (found).

(Ethyl-N-phenyl-P-ethenyl-phosphonamidate)-S-glutathion conjugate

To a capped vial Ethyl N-phenyl-P-ethynyl-phosphonamidate (31 mg, 0.15mmol) and potassium carbonate (7 mg, 0.05 mmol) was added. The mixturewas suspended in a 1 to 1 mixture of DMF/H₂O (500 μL). Then(2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}-butanoicacid (31 mg, 0.1 mmol) was added and the mixture was stirred at roomtemperature for 3 hours. Afterwards the mixture was diluted with H₂O (5mL) and CH₂Cl₂ (5 mL) was added. After extraction the phases wereseparated and the organic layer was extracted three times with H₂O (5mL). The aqueous layers were washed three times with CH₂Cl₂ (5 mL).Afterwards the solvent was removed under reduced pressure. The crudemixture was purified by preparative HPLC eluting with acetonitrile andammonium acetate buffer. The product was obtained as ammonium acetatesalt in a yield of 35.5 mg (0.061 mmol, 61%). ¹H NMR (300 MHz, DeuteriumOxide): 5=7.37 (d, J=12.6 Hz, 1H, S—CH, Z), 7.21 (t, J=7.9 Hz, 2H, ArH),6.94 (dd, J=7.8, 5.3 Hz, 3H, ArH), 5.77 (dd, J=17.5, 12.2 Hz, 1H, PCH,Z), 4.45 (ddd, J=12.8, 8.3, 5.2 Hz, 1H), 4.01 (q, J=7.4 Hz, 2H, CH₂),3.84-3.52 (m, 2H, CH₂), 3.21 (dd, J=14.7, 5.1 Hz, 1H, CH), 3.02 (dd,J=14.6, 8.5 Hz, 1H, CH), 2.45-2.20 (m, 2H, CH₂), 1.96 (q, J=7.2 Hz, 2H,CH₂), 1.19 (t, J=7.1 Hz, 3H, CH₃) ppm. ¹³C NMR: (75 MHz, DeuteriumOxide) δ=174.66, 174.45, 173.59, 171.25, 171.19, 151.73, 139.17, 129.38,122.06, 117.75 (d, J=6.8 Hz), 86.89, 86.83, 86.72, 62.14, 53.76 (d,J=12.1 Hz), 42.35, 35.94, 31.18, 25.92, 15.41 ppm. ³¹P NMR: (122 MHz,Deuterium Oxide) δ=18.96, 18.11 (d, J=4.2 Hz) ppm. HRMS ESI-TOF m/z[M+H]⁺=517.1516 (calcd.); 517.1526 (found).

Thiol-Addition of Ethanethiol and Glutathione to Alkynyl-Phosphonamidate

Ethanethiol was chosen as aliphatic model substrate. All experimentswere conducted for 3 hours at room temperature in 0.1 mmol scale using400 μL of the solvent. Conversions and diastereoselectivities weredetermined by ³¹P-NMR and ¹H-NMR (Scheme 4).

First experiments confirmed the formation of both the E- and theZ-conformational isomer. The vicinal H—H coupling constant of 12.5 Hz ofthe major diastereomer and 21.7 Hz of the minor diastereomer in the ¹HNMR of the diastereomeric mixture indicates that the Z-isomer is themajor product for all the reaction conditions (see Table 3).

TABLE 3 Screening of solvents for the base mediated hydrothiolation ofelectron-deficient alkynyl phosphonamidates. Entry Solvent BaseConversion E/Z 1 CH₂Cl₂ K₂CO₃ 100% 1:99 2 EtOH K₂CO₃ 100% 2:98 3 DMF/H₂OK₂CO₃ 100% 2:98 (1:1) 4 THF K₂CO₃ 100% 2:98 5 DMF K₂CO₃ 100% 5:95 6 DMSOK₂CO₃ 100% 12:88 

The influence of the solvent to the thiol-addition was then furtherinvestigated revealing quantitative formation of the thiol adduct forevery tested solvent. Full conversion was achieved in all of the testedsolvents. DMSO showed the lowest diastereoselectivity (12% E-product).Therefore the influence of the base was than further investigated inDMSO and in DMF/H₂O (1:1) (Table 4).

TABLE 4 Screening of bases for the hydrothiolation of electron-deficientalkynyl phosphonamidates in DMSO and DMF/H₂O (1:1). DMSO DMF/H₂O EntryBase Conversion E/Z Conversion E/Z 1 Et₃N  5% —/—  36% 3:97 2 (NH₄)₂CO₃100% 4:96 100% 2:98 3 Na₂CO₃ 100% 6:94 100% 1:99 4 Rb₂CO₃ 100% 8:92 100%2:98 5 K₂CO₃ 100% 12:88  100% 2:98 6 Cs₂CO₃ 100% 17:83  100% 2:98

It turned out that the diastereoselectivity of the reaction in DMSO isdependent of the applied base. In contrast to this, reactions in aqueoussystems always delivered the Z-alkene as the major product.

In conclusion it was possible to optimize the reaction conditions of themodel reactions. The reaction can be applied in aqueous solvent systemsand quantitative conversions can be achieved at room temperature after 3hours using mild carbonate bases. No side reactions were observed.

In the next step these optimized reaction conditions were now applied tosynthesize a water soluble glutathion phosphonamidate conjugate (Scheme5).

The conjugate could be isolated by semipreparative HPLC under basicconditions as a diastereomeric mixture in a yield of 61%. Havingreasonable quantities of this water soluble phosphonamidate-conjugate inhand, studies could be performed in order to determine the hydrolyticproperties of the phosphorus-nitrogen bond. For these studies a 3 μMsolution of conjugate and the standard tetramethylphosphonium bromide(1.2 μM) in aqeuous buffer was prepared and the hydrolysis of thephosphonamidate was characterized by monitoring the decay of theconjugate against the standard by means of ³¹P NMR over 24 hours. Theresults are shown in FIG. 1 which shows the hydrolytic decay of theGSH-phosphonamidate conjugate under acidic conditions.

Under strong acidic conditions (1 M HCL, pH 0.36) the phosphonamidateshowed rapid decomposition, which is represented by the lower curve(circles). For slight acidic conditions (150 mM NH₄OAc-buffer, pH=4.76),as depicted in the blue curve the compound was stable over the durationof the measurement (squares).

The rate of the reaction was determined by HPLC. Glutathione was addedto a solution of ethyl-N-phenyl alkynyl phosphonamidate in aqueousbuffer at slightly basic pH. The reaction was stopped after several timepoints by the addition of an acidic buffer and analyzed by HPLC-UV,referring to inosine as an internal standard. FIG. 2 refers to theconsumption of ethyl-N-phenyl alkynyl phosphonamidate in the reactionwith glutathione at pH 8.5. HPLC UV traces were taken at different timepoints. Experiment was performed in triplicate.

As FIG. 2 shows, we achieve a very fast conversion of more than 95% ofthe alkynyl phosphonamidate starting material after 15 min at pH 8.5.

Staudinger-Induced Thiol-Addition of RGD Peptides to GFP

In a next proof of principle study we synthesized an azido-containingcyclic RGD peptide (c(RGDfK)), which is known to bind to overexpressedintegrins in cancer cells. This cyclic azido-peptide was reacted withthe bisethoxyalkyne-phosphonite to form the highly reactivephosphonamidate in 53% isolated yield after HPLC with no observedby-product formation (Scheme 6, which is depicted in FIG. 40 ).

Scheme 6, which is depicted in FIG. 40 , shows the Staudinger-inducedthiol-addition of cyclic azido-RGD-peptides to GSH.

Synthesis of c(RGDfK)-azide

The cyclic RGDfK-azido peptide was synthesized manually on a NovaSynTGTalcohol resin with a loading of 0.26 mmol/g. First the resin wasactivated by stirring 480.7 mg resin in 2.5 ml toluene and 480 μlacetylchloride at 60° C. for three hours. Double coupling ofFmoc-Asp(OAII)-OH (123.56 mg, 0.3125 mmol, 2.5 eq) was performed in DCMusing DIPEA (212.6 μl, 1.25 mmol, 10 eq.) as activating base each forone hour. Further amino acid couplings were performed by mixing aminoacid (0.25 mmol, 2 eq.), HATU (0.25 mmol, 2 eq.) and DIPEA (0.5 mmol, 4eq.) in DMF and coupling once for 30 minutes and once for one hour. Fmocdeprotection was accomplished with 20% piperidine in DMF. After thefinal amino acid coupling the alloc deprotection was achieved bytreating the resin with Pd(P(Ph₃)₄) (433 mg, 0.375 mmol, 3 eq.) inchloroform/acetic acid/NMM (37:2:1; v:v:v) for two hours in an argonatmosphere, followed by Fmoc deprotection and cyclisation with HATU(0.25 mmol, 2 eq.) and DIPEA (0.5 mmol, 4 eq.) in DMF for 16 hours. Tobe abled to install the aromatic azide on the lysine residueFmoc-Lys(dde)-OH was used in the solid phase synthesis and wasorthogonally deprotected on resin using 2% hydrazine in DMF three timesfor three minutes, followed by coupling of 4-azidobenzoic acid (81.65mg, 0.5 mmol, 4 eq.) with HATU (190 mg, 0.5 mmol, 4 eq.) and DIPEA (1mmol, 8 eq.) in DMF for two hours. Cleavage from the resin was performedusing TFA/DCM (75:25; v:v) for 2.5 hours. Precipitation was carried outin cold and dry ether. The crude was analyzed by UPLC-MS and either usedas crude in the following staudinger reaction or purified by preparativereverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA);5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). The product wasgained as white powder (8.0 mg, 11.0 μmol, 8.5% yield) and was analyzedby analytical UPLC (5 to 95% of acetonitrile in water containing 0.1%TFA on a RP-C18 column). The UPLC chromatogram of the c(RGDfK)-azide isshown in FIG. 7 . LRMS: m/z: 749.67 [M+H]⁺ (calcd. m/z: 749.3485).

Synthesis of c(RGDfK)-phosphonamidate Alkyne Bisethoxyalkyne-phosphoniteSynthesis

Ethynyl magnesium bromide in THF (5 M, 2 ml, 1 mmol, 1 eq.) was cooledto −78° C. in a flame dried schlenk flask and diethylchlorophosphate(0.143 ml, 1 mmol, 1 eq.) was added. The solution was stirred for 10minutes at −78° C. and let warm to room temperature and stirred foranother 90 minutes. The full consumption of starting material waschecked by ³¹P-NMR (product at 126.73 ppm; see FIG. 6 : CrudeBisethoxyalkyne-phosphonite synthesis to FIG. 9 ) and used as crude inthe following staudinger reaction with azido-c(RGDfK).

Staudinger Reaction on c(RGDfK)-azide

When crude peptide was used it (66 mg, 88.2 μmol, 1 eq.) was dissolvedin DMSO (4 ml, 22 mM) and dried in a flame dried flask for one hourprior to adding bisethoxyalkyne-phosphonite (volume according topercentage of product determined by NMR, 132.3 μmol, 1.5 eq.). After thereaction mixture was stirred over night at room temperature 4 ml ofwater were added and stirred for 6 hours, before lyophilization. Thecrude product was purified by semi-preparative reverse phase C18 HPLC(0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water(0.1% TFA)/MeCN (0.1% TFA)) and gave the product as a white powder (6.2mg, 6.64 μmol, 5.3% overall yield).

Using the purified c(RGDfK)-azido peptide (6.9 mg, 9.14 μmol, 1 eq.) itwas dissolved in DMSO (1.5 ml, 6 mM) and dried in a flame dried flaskfor one hour prior to adding bisethoxyalkyne-phosphonite (volumeaccording to percentage of product determined by NMR, 36.56 μmol, 4eq.). After the reaction mixture was stirred over night at roomtemperature 1.5 ml water was added and stirred again for six hoursbefore lyophilization. The crude product was purified bysemi-preparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1%TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA))and gave the product as a white powder (4.1 mg, 4.89 μmol, 53.5% yield).

The final product was analyzed by LC-UV: rt. 5.0 min (0-1 min 95/5,water (0.1% TFA)/MeCN (0.1% TFA); 1-16.5 min 5/95, water (0.1% TFA)/MeCN(0.1% TFA) on RP-C18 column) and mass. The chromatogram of thec(RGDfK)-alkyne is shown in FIG. 8 . HRMS: m/z: 839.3636 [M+H]⁺ (calcd.m/z: 839.3606)

Hydrothiolations of Electron-Deficient c(RGDfK)-phosphonamidate AlkyneModel Reaction with Glutathione

Glutathione (1 mg, 3.25 μmol, 1 eq.) and c(RGDfK)-phosphonamidate alkyne(1.24 mg, 3.25 μmol, 1 eq.) were mixed in 135 μl 10 mMammoniumbicarbonate buffer pH 9.2 and 15 μl acetonitrile (c=21.6 mM).After 10 minutes of shaking quantitative conversion to the additionproduct was observed by LC-UV/MS.

The final product was analyzed by LC-UV: rt. 4.3/4.4 min (0-1 min 95/5,water (0.1% TFA)/MeCN (0.1% TFA); 1-16.5 min 5/95, water (0.1% TFA)/MeCN(0.1% TFA) on RP-C18 column)

HRMS: m/z: 1146.4451 [M+H]⁺ (calcd. m/z: 1146.4444), 573.7321 [M+2H]²⁺(calcd. m/z: 573.7262)

As a first test substrate for the reaction with thiols, we usedglutathione (GSH) and found a fast and high yielding addition of thethiol to the phosphonamidate-alkyne in nearly quantitative conversionsafter 10 minutes under slightly basic conditions at pH 8.8 at roomtemperature (FIG. 3 : Staudinger-induced thiol-addition of cyclicazido-RGD-peptides to GSH).

On this model addition product we conducted stability studies atdifferent pH and under the addition of thiols like MesNa and DTT atneutral and basic pH. It could have been shown that the formed productis stable in a broad pH range from pH 2.3 until pH 9.0 (FIG. 4 : pHstability of c(RGDfK)-Glutathion).

Also the product is stable towards high concentration (0.2 M, 100 eq.)of DTT and MesNa at physiological pH (PBS buffer; pH 7.4). At pH 9.0MesNa is slowly adding to the formed double bond (10% addition productformed after four days). In contrast to that DTT is rapidly forming anaddition product with (42% after 30 hours) and followed by degradationover time.

Staudinger-Induced Thiol-Addition of GFP Protein

In the next step we probed the Staudinger-induced conjugation reactionwith a Cys-containing model protein. Here we used a mutated eGFP bearingonly one addressable cysteine for the thiol conjugation to the cyclicRGD-phosphonamidate.

Reaction with GFP C70M S147C

GFP C70M S147C (3.13 nmol, 1 eq) was rebuffered to 100 μl 10 mMAmmoniumbicarbonte pH 8.4 and c(RGDfK)-phosphonamidate alkyne (0.08 mg,93.9 nmol, 30 eq.) was added. The reaction mixture was shaken at 37° C.and 800 rpm for three hours. Finally the mixture was spin filtratedusing Amicon Spin filters with a 10 kDa MWCO. After spinfiltrating thesample ten times at 14000 rpm for 5 minutes and adding fresh 10 mMAmmoniumbicarbonate buffer MALDI-TOF analysis was conducted and verifiedtotal conversion of GFP C70M S147C to the desired product. The structureof the product is shown in FIG. 30 .

MALDI TOF: expected (in Da): 28605.31 (M+H⁺), 14303.16 (M+2H⁺); found(in Da): 28608.46 (M+H⁺), 14294.46 (M+2H⁺)

With this approach we could validate the feasibility of this reaction onthe protein level at a concentration of 31 μM, in which the conjugatewas formed again in virtually quantitative conversions, as verified byMALDI-MS analysis and MS/MS analysis of the digested protein conjugate(FIG. 5 : Staudinger-induced thiol-addition to thiol-containing eGFP.).

Stability Studies for c(RGDfK)-glutathion

c(RGDfK)-glutathion was solved at a concentration of 2 mM in differentsolvents (0.1 M HCl at pH 1; 30% acetonitrile in water containing 0.1%TFA with a pH of 2.3; PBS buffer rat pH 7.4; ammonium acetate buffer ratpH 9.0; 0.05 M NaOH at pH 12) and 0.5 mM of Inosine was added asinternal standard. The stability of the starting material was thenmonitored over three days.

The stability studies in presence of a competing thiolc(RGDfK)-glutathion was solved in either PBS or 1 M Tris HCl pH 9.0 at aconcentration of 2 mM and 10 eq. DTT or MesNa was added. The mixture wasmonitored over several days.

Antibody Conjugation with Alkyne Phosphonamidates

First experiments were conducted with Cetuximab, a monoclonal IgG1antibody against human epidermal growth factor. The antibody wasmodified with a biotin phosphonamidate and analyzed by SDS-PAGE undernon reducing conditions, followed by anti-biotin western blotting(Scheme 7, which is depicted in FIG. 41 ).

Scheme 7, which is depicted in FIG. 41 , shows the two-step reductionand alkylation approach for cysteine selective antibody modificationwith a biotin modified alkynyl phosphonamidate.

The intact antibody was reduced by incubation with DTT in 50 mM boratecontaining PBS (pH 8.0) at 37° C. Excess of DTT was removed after thereaction by size exclusion columns and the reduced antibody fragmentswere incubated with a biotin phosphonamidate (1.1 equivalents per thiol)in 50 mM ammonium bicarbonate buffer (pH 8.5). EDTA (1 mM) was added tothe reaction mixture to complex heavy metal ions that promote disulfideformation.

Western blot analysis confirmed modification of the antibody fragments,even though the intact antibody is not formed back by reoxidation of theremaining cysteins. This could be explained by a high degree ofmodification. No modification could be detected without prior reductionof the disulfide bonds. Thus further confirming the high selectivity ofthese compounds for free cysteine residues. Further experiments willinclude the determination of the degree of modification and experimentsthat prove the functionality of the modified antibody (see FIG. 10 :Western blot analysis after non reducing SDS-PAGE. SM: Cetuximabstarting material. 1:5 min, 2:1 h, 3:2 h, 4:20 h incubation with abiotin modified phosphonamidate. Reaction with (left) and without(right) prior reduction of the disulfides).

Cysteine selective modification was further confirmed by tryptic digestof the cetuximab phosphonamidate conjugates, followed by MS/MS analysis.To simplify the MS/MS spectra, the modification was conducted underpreviously described conditions with the structurally simplerethyl-N-phenyl alkynyl phosphonamidate. Modification of Cys 263 of theheavy chain and Cys 214 of the light chain could be confirmed by MS/MS(HCD fragmentation) while no modification was detected without priorreduction of the disulfide bonds.

Staudinger-Induced Thiol-Additions with Vinyl Phosphonites a) Synthesisof Various Borane Protected Vinyl Phosphonites

Diethyl vinylphosphonite was synthesized based on previously publishedprotocols by alkylation of diethyl chlorophosphite with vinylmagnesiumbromide and subsequent borane addition (13) (Scheme 8). The desiredphosphonite was isolated in 37% yield.

Vinylphosphonites with different O-substituents were synthesizedstarting from phosphorous trichloride by substitution of two chlorideswith the corresponding alcohols in the presence of pyridine. The formedmono chloro phosphite was reacted with the vinyl Grignard reagent andprotected with borane. All these steps were performed in a one-potstrategy.

As some alcohols are not compatible with subsequent addition of theGrignard reagent we applied an alternative route to the synthesis ofphosphonites derived from these alcohols, starting frombis(diisopropylamino)chlorophosphine. alkylation tobis(diisopropylamino)vinylphosphine in the first step enabled tetrazolemediated addition of the alcohol in more polar solvents likeacetonitrile in the second step. All phosphonites were treated withborane in situ and isolated by flash chromatography.

Experimental Part for IIa Diethyl Vinylphosphonite Borane

A 25-ml Schlenk flask was charged with 2.14 ml vinylmagnesium bromide(0.7 M in THF, 1.50 mmol, 1.5 eq.) under an argon atmosphere, cooled to−78° C. and 140 μl diethyl chlorophosphite (1.00 mmol, 1.0 eq.) wereadded drop wise. The yellowish solution was allowed to warm to 0° C.,stirred for another two hours and 1.00 ml of Borane (1.0 M in THF, 1.00mmol, 1.0 eq.) were added and stirred for one more hour at 0° C. Theorganic solvents were removed under reduced pressure and the crudeproduct was purified by flash column chromatography on silica gel(Hexane/EtOAc, 9:1) to yield the desired compound as colourless oil. (60mg, 0.37 mmol, 37.0%)

¹H NMR (300 MHz, Chloroform-d) δ=6.36-6.03 (m, 3H), 4.19-3.96 (m, 4H),1.33 (t, J=7.1, 6H), 0.55 (ddd, J=190.3, 94.1, 16.6, 3H). ¹³C NMR (75MHz, Chloroform-d) δ=134.62 (d, J=8.7), 130.12 (d, J=75.0), 63.16 (d,J=4.8), 16.59 (d, J=5.6). ³¹P NMR (122 MHz, Chloroform-d) δ=129.58 (dd,J=167.1, 82.6).

NMR data is in accordance with those reported in the literature.¹⁸

Di(2-nitrobenzyl) Vinylphosphonite Borane

The compound was synthesized according to the general procedure A fromPCI₃ (260 μl, 3.00 mmol). The pure borane protected phosphonite waspurified by flash column chromatography (Hexane/EtOAc, 4:1) and obtainedas a yellowish solid. (555 mg, 1.48 mmol, 49.2%)

¹H NMR (300 MHz, Chloroform-d) δ=8.10 (d, J=8.2, 2H), 7.77-7.63 (m, 4H),7.57-7.44 (m, 2H), 6.51-6.18 (m, 3H), 5.45 (qd, J=14.8, 7.5, 4H),1.42-−0.02 (m, 3H). ¹³C NMR (75 MHz, Chloroform-d) δ=146.80, 136.84 (d,J=10.2), 132.52 (d, J=6.8), 129.10, 129.05, 128.67, 128.61 (d, J=74.3),125.09, 65.56 (d, J=3.6). ³¹P NMR (122 MHz, Chloroform-d) δ=136.23 (dd,J=151.3, 56.0). HRMS for C₁₆H₁₈BN₂NaO₆P⁺ [M+Na]⁺ calcd: 399.0888, found:399.0885

Di(2-(2-methoxyethoxy)ethyl) Vinylphosphonite Borane

The compound was synthesized according to the general procedure A fromPCI₃ (130 μl, 1.50 mmol). The pure borane protected phosphonite waspurified by flash column chromatography (CH₂Cl₂/MeOH, 19:1 to 9:1) andobtained as a colourless oil. (34 mg, 0.11 mmol, 7.3%)

¹H NMR (300 MHz, Chloroform-d) δ=6.37-6.00 (m, 3H), 4.16 (dq, J=7.6,4.9, 4H), 3.70 (t, J=4.8, 4H), 3.64 (dd, J=5.8, 3.3, 4H), 3.54 (dd,J=5.9, 3.3, 4H), 3.38 (s, 6H), 1.14-−0.12 (m, 3H). ¹³C NMR (75 MHz,Chloroform-d) δ=135.00 (d, J=8.9), 129.51 (d, J=75.7), 71.79, 70.47,70.21 (d, J=6.0), 65.92 (d, J=5.2), 58.95. ³¹P NMR (122 MHz,Chloroform-d) δ=133.77-130.56 (m).

Diphenyl Vinylphosphonite Borane

The compound was synthesized according to the general procedure A fromPCI₃ (393 μl, 4.50 mmol). The pure borane protected phosphonite waspurified by flash column chromatography (Hexane/EtOAc, 4:1) and obtainedas a colourless oil. (700 mg, 2.71 mmol, 60.3%)

¹H NMR (300 MHz, Chloroform-d) δ=7.39 (td, J=7.7, 5.5, 4H), 7.30-7.17(m, 6H), 6.67-6.18 (m, 3H), 1.48-0.01 (m, 3H). ¹³C NMR (75 MHz,Chloroform-d) δ=151.27 (d, J=8.7), 137.01 (d, J=12.5), 129.70 (d,J=1.0), 129.05 (d, J=71.1), 125.35 (d, J=1.3), 120.90 (d, J=4.2). ³¹PNMR (122 MHz, Chloroform-d) δ=134.08-130.87 (m). HRMS for C₁₄H₁₆BNaO₂P⁺[M+Na]⁺ calcd: 281.0873, found: 281.0873.

Bis(4-(2-nitro-5-(oxypropargyl)benzyloxy)phenyl) Vinyl PhosphoniteBorane

The compound was synthesized according to the general procedure B fromBis(diisopropylamino)chlorophosphine (71 mg, 0.27 mmol). The pure boraneprotected phosphonite was purified by flash column chromatography(Hexane/CH₂Cl₂, 1:1) and obtained as a yellowish solid. (75 mg, 0.11mmol, 41.9%)

¹H NMR (300 MHz, Chloroform-d) δ=8.25 (d, J=9.1, 2H), 7.47 (d, J=2.8,2H), 7.17-6.87 (m, 10H), 6.62-6.14 (m, 3H), 5.48 (s, 4H), 4.78 (d,J=2.4, 4H), 2.55 (t, J=2.4, 2H), 1.51-−0.24 (m, 3H). ¹³C NMR (75 MHz,Chloroform-d) δ=161.89, 155.27 (d, J=1.3), 145.34 (d, J=8.4), 140.04,137.08 (d, J=12.6), 136.97, 129.06 (d, J=71.3), 127.77, 121.93 (d,J=4.0), 115.74 (d, J=1.1), 113.85, 113.79, 76.89, 76.89, 67.37, 56.24.³¹P NMR (122 MHz, Chloroform-d) δ=136.36-131.69 (m). HRMS forC₈₄H₃₀BN₂NaO₁₀P⁺ [M+Na]⁺ calcd: 691.1623, found: 691.1629.

Bis(2-nitro-5-(oxypropargyl)benzyl) Vinyl Phosphonite Borane

The compound was synthesized according to the general procedure B fromBis(diisopropylamino)chlorophosphine (513 mg, 1.92 mmol). The pureborane protected phosphonite was purified by flash column chromatography(Hexane/CH₂Cl₂, 4:1) and obtained as a yellowish solid. (704 mg, 1.45mmol, 75.6%)

¹H NMR (300 MHz, Chloroform-d) δ=8.19 (d, J=9.1, 2H), 7.31 (d, J=2.8,2H), 7.00 (dd, J=9.2, 2.8, 2H), 6.58-6.22 (m, 3H), 5.49 (qd, J=15.5,7.4, 4H), 4.81 (d, J=2.4, 4H), 2.62 (t, J=2.4, 2H), 1.31-0.12 (m, 3H).¹³C NMR (75 MHz, Chloroform-d) δ=161.86, 139.86, 136.92 (d, J=10.4),135.78 (d, J=6.8), 128.43 (d, J=73.2), 127.79, 114.10, 113.92, 76.95,76.89, 65.54 (d, J=3.6), 56.31. ³¹P NMR (122 MHz, Chloroform-d) δ=136.32(d, J=107.0). HRMS for C₂₂H₂₂BN₂NaO₈P⁺ calcd: 507.1099, found: 507.1111.

Bis(2,2,2-trifluoroethyl) Vinyl Phosphonite Borane

The compound was synthesized according to the general procedure B fromBis(diisopropylamino)chlorophosphine (266 mg, 1.00 mmol). The pureborane protected phosphonite was purified by flash column chromatography(Hexane/CH₂Cl₂, 9:1 to 4:1) and obtained as a colourless liquid. (87 mg,0.32 mmol, 32.2%) ¹H NMR (300 MHz, Chloroform-d) δ=6.52-6.11 (m, 3H),4.36 (p, J=8.1, 4H), 0.62 (ddd, J=203.0, 103.5, 15.0, 3H). ¹³C NMR (75MHz, Chloroform-d) δ=137.57, 127.48 (d, J=79.1), 122.37 (qd, J=276.0,7.5), 63.60 (qd, J=37.7, 2.5). ¹⁹F NMR (282 MHz, Chloroform-d) δ=2.13.³¹P NMR (122 MHz, Chloroform-d) δ=145.49 (dd, J=135.6, 65.1).

Bis-(4-Hydroxyphenyl) Vinyl Phosphonite Borane

The compound was synthesized according to the general procedure B fromBis(diisopropylamino)chlorophosphine (534 mg, 2.00 mmol) and Hydrochinon(2.20 g, 10 eq.). The pure borane protected phosphonite was purified byflash column chromatography (Hexane/EtOAc, 4:1 to 1:1) and obtained as acolourless solid. (280 mg, 0.96 mmol, 48.3%)

¹H NMR (300 MHz, DMSO-d₆) δ=9.49 (s, 2H), 6.96 (d, J=8.5, 4H), 6.74 (d,J=8.9, 4H), 6.63-6.22 (m, 3H), 1.20-−0.12 (m, 3H). ³¹P NMR (122 MHz,DMSO-d₆) δ=131.80. HRMS for C₁₄H₁₆BNaO₄P⁺ calcd: 313.0771, found:313.0774.

Di(4-nitrobenzyl) Vinylphosphonite Borane

The compound was synthesized according to the general procedure B fromBis(diisopropylamino)chlorophosphine (533 mg, 2.00 mmol). The pureborane protected phosphonite was purified by flash column chromatography(Hexane/CH₂Cl₂, 9:1 to 4:1) and obtained as a white solid. (540 mg, 1.44mmol, 71.8%)

¹H NMR (300 MHz, Chloroform-d) δ=8.17 (d, J=8.6, 4H), 7.49 (d, J=8.5,4H), 6.47-6.16 (m, 3H), 5.12 (qd, J=13.2, 8.3, 4H), 1.40-−0.00 (m, 3H).¹³C NMR (75 MHz, Chloroform-d) δ=147.66, 143.09 (d, J=6.1), 136.50 (d,J=10.2), 128.80 (d, J=76.0), 127.78, 123.73, 67.30 (d, J=3.8). ³¹P NMR(122 MHz, Chloroform-d) δ=137.95 (d, J=95.6).

2-nitro-5-(oxypropargyl)benzyl Alcohol

A 5 ml-microwave tube was charged with 200 mg of 5-Hydroxy-2-nitrobenzylalcohol (1.18 mmol, 1.0 eq.), 245 mg K₂CO₃ (1.77 mmol, 1.5 eq.), 132 μlPropargyl bromide (80 wt. % solution in Toluene) and 4 ml DMF. Theresulting suspension was irradiated for 1 h at 100° C. After cooling toroom temperature, 5 ml of water were added. The resulting precipitatewas filtered, washed three times with water and vacuum dried to give 179mg of light brown solid. (0.87 mmol, 73.2%) NMR data is in accordancewith those reported in the literature.¹⁹

4-(2-nitro-5-(oxypropargyl)benzyloxy)phenol

A flame dried Schlenk-tube, 400 mg of 2-nitro-5-(oxypropargyl)benzylalcohol (1.93 mmol, 1.0 eq.), together with 850 mg hydrochinon (7.72mmol, 4.0 eq.) and 750 mg of triphenylphosphine (2.90 mmol, 1.5 eq.)were dissolved in 10 ml of dry THF. The solution was cooled to 0° C. and1.33 ml of diethyl azodicarboxylate (40% solution in Toluene) (2.90mmol, 1.5 eq.) were added dropwise and the reaction was allowed to warmto room temperature overnight. The crude product was dry packed on asilica column for purification, eluting with Hexan/EtOAc (7:3 to 3:2),yielding 505 mg of a yellow solid. (1.68 mmon, 87.5%)

¹H NMR (300 MHz, Chloroform-d) δ=8.26 (d, J=9.1, 1H), 7.51 (d, J=2.8,1H), 7.00 (dd, J=9.2, 2.9, 1H), 6.89 (d, J=9.0, 2H), 6.80 (d, J=9.0,2H), 5.46 (s, 2H), 4.79 (d, J=2.4, 2H), 2.54 (t, J=2.4, 1H). ¹³C NMR (75MHz, Chloroform-d) δ=161.86, 152.11, 150.03, 140.10, 137.71, 127.69,116.11, 116.03, 113.88, 113.69, 76.95, 76.68, 67.66, 56.20. HRMS forC₁₆H₁₄NO₅ ⁺ [M+H]⁺ calcd: 300.0866, found: 300.0871.

Small Molecule Studies: Reaction of Unprotected Alkene Phosphonites withDifferent Azides

The Staudinger phosphonite reaction with vinyl phosphonites was firstinvestigated with rather simple diethyl derivatives. Those weresynthesized by alkylation of commercial available diethylchlorophosphite and reacted with different aliphatic and aromatic azidesin situ. The desired phosphonamidates were isolated by columnchromatography, after Hydrolysis of the Phosphonamidates.

Varying the O-substituents of the Phosphonamidates allows the finetuning of the reactivity in the thiol-addition as well as theinstallation of a third functionality to the system. Phosphonamidateswith substituents other than ethyl were synthesized by staudingerphosphonite reaction of the respective phosphonites. Isolated boraneprotected phosphonites were treated with DABCO to form the reactiveP(III) species and reacted with an azide in situ to form thephosphonimidate. Subsequent Hydrolysis by water addition formed thedesired phosphonamidates in moderate yields.

The 2-nitro-benzyl group is widely known as a photolabile substituentand has been shown to release attached molecules upon UV-irradiation.²⁰From our expertise in phosphonamidate chemistry, we knew that theP—N-Bond of the phosphonamidate the very labile once the Phosphonamidateester is cleaved. Therefore we wanted to synthesize 2-nitro-benzylsubstituted Phosphonamidates that enable the controlled light mediatedrelease of an amine from the thiol addition conjugates.

Several 2-nitro-benzyl substituted phosphonamidates could besynthesized, including a photo cleavable biotin as well as a Cy5-dye anda DABCYL quencher variant. An additional alkyne at the 2-nitro-benzylgroup enables the installation of a third functionality to the systemwire copper catalyzed click chemistry, which can be cleaved of again byphoto irradiation.

Further fine tuning of the reactivity of the subsequent thiol-additionwas achieved by changing the electronic properties of thephosphonamidates. Therefore different phenyl- as well as trifluoroethylderivatives were synthesized.

Some phosphonites could not be isolated with a borane protection group,as their corresponding alcohols are not compatible with borane addition.We were able to show that these phosphonites can be used in an in situsynthesis with an azide without isolation of the phosphonite as depictedin Scheme 16.

Stability of a phosphonamidate to different pHs was proven by ³¹P-NMR inaqueous buffers at room temperature. In a first experiment,Ethyl-N-phenyl-P-vinyl-phosphonamidate was chosen to measure stability.It turned out that the compound is stable over a broad pH range.P—N-bond cleavage occurred under strong acidic conditions (FIG. 11 :Stability of Ethyl-N-phenyl-P-vinyl-phosphonamidate to different pHsover time).

Thiol-Addition of Small Molecule Thiols and Glutathione to VinylPhosphonamidates

In a first study, vinyl phosphonamidates were reacted with differentsmall molecule thiols under reaction conditions that previously workedwell for alkynyl phosphonamidates. Full conversion of thePhosphonamidate starting material could be observed after 3 h treatmentwith one equivalent of a thiol in presence of potassium carbonate.

In the next step these reaction conditions were now applied tosynthesize water soluble glutathione phosphonamidate conjugates. Thereaction proceeded in case of the water soluble4-carboxyphenyl-phosphonamidate without the addition of any organicsolvent. The highly polar products were isolated by semi preparativeHPLC with a slightly basic gradient.

Stability of a thiol-adduct to different pHs was proven by ³¹P-NMR inaqueous buffers at room temperature. The conjugates showed excellentstability over a broad pH range. P—N-bond cleavage occurred under strongacidic conditions. An elimination of the thiol referred to as retrothiol-addition was not observed (FIG. 12 : Stability of a glutathionephosphonamidate adduct to different pHs over time).

The effect of the O-substituent on the reaction rate was investigated bythe addition of glutathione to a solution of various N-phenyl alkynylphosphonamidates in ammonium bicarbonate buffer at pH 8.5. Conversion ofdifferent phosphonamidates over time is shown in FIG. 11 .

We found out that that vinyl phosphonamidates are much slower in thereaction with thiols than there corresponding alkynyl derivatives. Weassumed that exchanging the electron donating ethyl group of thephosphonamidates to more electron withdrawing substituents shouldfurther increase the electrophilicity and therefore raise the rate ofthe thiol addition. Exchanging the ethyl to a phenyl group alreadyreduces the half-life time t_(1/2) of the staring material in thereaction from ten hours to one hour. Trifluoroethyl further reducest_(1/2) to thirty minutes while 2-nitro benzyl reacts to fifty percentin two hours (FIG. 13 : Consumption of various N-phenyl vinylphosphonamidates in the reaction with glutathione at pH 8.5. HPLC UVtraces were taken at different time points. Experiments were performedin triplicates).

Thiol-addition to vinyl phosphonamidates on protein level

First experiments with alkene phosphonamidates on protein level wereconducted with the water solubleEthyl-N-(4-carboxy-phenyl)-P-vinyl-phosphonamidate. As previous studiesindicated that carbonate bases work very well in the promotion of thethiol-addition, ammonium bicarbonate buffer at pH 9.0 was chosen for thefirst experiments. A mutated eGFP variant bearing only one addressablecysteine was selected for the study.

Scheme 19, which is depicted in FIG. 31 , shows an addition of a watersoluble vinyl phosphonamidate to eGFP with one addressable cysteine.

The protein was incubated with 50 equivalents of the phosphonamidate at37° C. Even though MALDI/MS analysis of the reaction mixture after 16hours showed still unreacted protein, we were very pleased to observeformation of the desired protein conjugate.

Further eGFP conjugation experiments were conducted with a fluorescentCy5-Phosphonamidate and observed by in-gel fluorescence measurements ofthe Cy5-channel.

Scheme 20, which is depicted in FIG. 32 , shows a Cy5 phosphonamidatelabeling of eGFP with one addressable cysteine. In gel fluorescence readout after SDS Page confirms selective Cy5 labeling.

The selectivity of the reaction for cysteine residues could be confirmedin this experiment. Neither an eGFP variant without any accessiblecysteine incubated with the phosphonamidate nor addition of a Cy5 azideto the Cys containing eGFP showed fluorescent labeling. Addition of 5%DMSO (line 1) or acetonitrile (line 3) to the reaction mixture were bothsufficient in solubilizing the dye without influencing the reactionitself.

Light Cleavable Triple Conjugation

We mentioned earlier that we were able to synthesize phosphonamidateswith o-nitro benzyl substituents bearing an additional alkyne handle forCuAAC. One possible application for these compounds is the installationof a biotin to the alkyne to purify protein conjugates. We envision thatthe biotin binds to streptavidin beads, unbound material can be washedaway and pure protein can be eluted by light irradiation.

In a first experiment on protein level, a simple N-phenylPhosphonamidate with an 0-substituted light cleavable Alkyne was reactedfirst with our single cysteine containing eGFP under previouslyoptimized conditions. After the step, an azido modified biotin wasattached to the construct via CuACC and the conjugates were analyzed byanti-biotin western blotting.

Scheme 21, which is depicted in FIG. 33 , shows a photocleavable alkynelabeling of eGFP with one addressable cysteine with subsequent biotinlabeling via CuACC and western blot analysis.

Western blot analysis confirmed successful conjugation of the biotin tothe eGFP construct. When eGFP without attached phosphonamidate was usedin the CuACC reaction no biotin was detected. The same is true in theabsence of Copper.

Further immobilization experiments on Streptavidin beads were conductedwith a phosphonamidate, Synthesized from an azido containing peptide anda single cysteine containing Ubiquitin. The high molecular weight of thepeptide induces a shift of the protein in the SDS gel, allowing theestimation of the conjugation yield.

Scheme 22, which is depicted in FIG. 34 , shows a photocleavable alkynelabeling of ubiquitin with one addressable cysteine with subsequentbiotin labeling via CuACC. Western blot analysis after immobilization onstreptavidin beads. 1: Ubiquitin starting material, 2: reaction mixtureafter CuACC, 3: Supernatant after incubation of the reaction mixturewith streptavidin agarose, 4: flow through after wash of streptavidinagarose, 5: boiled beads, 6: Irradiated beads.

The conjugation yield of the peptide to the protein could be estimatedto 60%. The final construct was successfully immobilized on streptavidinbeads. The constructs can be released by either boiling the beads in SDSbuffer, which releases the intact protein peptide conjugate orirradiation by UV light. The latter method cleaves the phosphonamidateester, leading to instability of the P—N-Bond and therefore release ofthe unconjugated protein.

Further experiments will be conducted with phosphonamidates that formintact esters upon light irradiation to release the conjugated constructupon light irradiation.

b) Antibody Conjugation with Vinyl Phosphonamidates

Vinyl phosphonamidates were also applied to the modification ofmonoclonal antibodies. As we found out that 2-nitro-benzyl substitutedvinyl phosphonamidates react faster in the thiol addition, we chosethose phosphonamidate with a biotin modification.

Scheme 23, which is depicted in FIG. 42 , shows the reduction ofantibody disulfides and subsequent modification with a biotin vinylphosphonamidate.

We chose the same reaction conditions for the reduction-alkylationprocedure as described previously with the exception of 4° C. for thethiol-addition, because we found out that lower temperatures slow downdisulfide formation and therefore lead to higher conjugation yield.

Western blot analysis confirmed cysteine selective modification of theantibody. High selectivity for free cysteine residues could be observedby the absence of a Signal in the anti-biotin western blot without priorreduction of the disulfide bonds. In contrast to the labelingexperiments with alkynyl phosphonamidates, this time reformation of theantibody fragments could be observed (FIG. 14 : Western blot analysisafter non reducing SDS-PAGE. SM: Cetuximab starting material. 1: 5 min,2: 1 h, 3: 2 h, 4: 4 h, 5: 20 h incubation with a biotin modifiedphosphonamidate. Reaction with (left) and without (right) priorreduction of the disulfides).

Cysteine selective modification was further confirmed by tryptic digestof the cetuximab phosphonamidate conjugates, followed by MS/MS analysis.To simplify the MS/MS spectra, the modification was conducted underpreviously described conditions with the structurally simplerphenyl-N-phenyl alkynyl phosphonamidate. Modification of Cys 264 and Cys146 of the heavy chain could be confirmed by MS/MS (HCD fragmentation)while no modification was detected without prior reduction of thedisulfide bonds.

Alkene Phosphonites in the Synthesis ASGP-R Addressing Drug Conjugates

We further want to apply our modular conjugation approach to thesynthesis of targeted drug conjugates. Khorev et al described previouslythe synthesis of an ASGP-R addressing trivalent ligand with a terminalamino modification. Based on this route, we synthesized the same ligandwith a terminal thiol modification (20).

Scheme 24, which is depicted in FIG. 43 , shows the synthesis of afluorescently labeled ASGP-R addressing Cy5 conjugate via the modularaddition to vinyl phosphonamidates.

Having the thiol modified, fully deprotected ligand in hand wesuccessfully conjugated the construct to our fluorescentCy5-phosphonamidate. With this conjugate, we can now monitor thesufficient uptake into hepatocytes by FACS analysis and fluorescentmicroscopy.

FIG. 15 shows the sequences mentioned throughout this description.

Introduction of the Alkyne-Phosphonamidate Moiety by Generic BuildingBlocks Via an Amide Bond

Generic building blocks as the amino-modified derivative N2 or theNHS-ester N1 shown in Scheme 25 can introduce an alkyne-phosphonamidatemoiety into functional molecules via amide bond forming reactions.

Scheme 25, which is depicted in FIG. 35 , shows alkyne-phosphonamidatesfor the chemoselective modification of Cys-residues. Introduction viachemoselective Staudinger-phosphonite reaction or amide coupling withthe generic building blocks N1 and N2.

This approach can be advantageous as one does not have to handle labileP(III) compounds. Furthermore, it has been shown that high yields can beachieved by using those generic building blocks, which is of aparticular interest for expensive starting materials.

Procedures for the Introduction of the Alkyne-Phosphonamidate Moiety byGeneric Building Blocks Via an Amide Bond Preparative HPLC

Preperative HPLC was performed on a Gilson PLC 2020 system (Gilson Inc,Wis., Middleton, USA) using a VP 250/32 Macherey-Nagel Nucleodur C18HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany). The followinggradients were used throughout all sections of this disclosure: MethodC: (A=H₂O+0.1% TFA (trifluoroacetic acid), B=MeCN (acetonitrile)++0.1%TFA, flow rate 30 ml/min, 5% B 0-5 min, 5-90% B 5-60 min, 90% B 60-65min. Method D: (A=H₂O+0.1% TFA, B=MeCN++0.1% TFA), flow rate 30 ml/min,5% B 0-5 min, 5-25% B 5-10 min, 25%-45% B 10-50 min, 45-90% 50-60 min,90% B 60-65 min.

Semi-Preperative HPLC

Semi-preparative HPLC was performed on a Shimadzu prominence HPLC system(Shimadzu Corp., Japan) with a CBM20A communication bus module, aFRC-10A fraction collector, 2 pumps LC-20AP, and a SPD-20A UV/VISdetector, using a VP250/21 Macherey-Nagel Nucleodur C18 HTec Spum column(Macherey-Nagel GmbH & Co. Kg, Germany). The following gradients wereused throughout all sections of this disclosure: Method E: (A=H₂O+0.1%TFA, B=MeCN++0.1% TFA), flow rate 10 ml/min, 5% B 0-5 min, 5-99% B 5-65min, 99% B 65-75 min.

General Procedure 1 for the Synthesis of Aromatic Azides

A 500-ml round-bottom flask was charged with 10 mmol aromatic amine,suspended in 15 ml water and cooled to 0° C. 5 ml of concentratedaqueous HCl were added, followed by drop-wise addition of 1.27 g sodiumnitrite (15.00 mmol, 1.50 eq.) solution in 10 ml Water. The mixture wasstirred for 20 min at 0° C., 100 ml EtOAc (ethyl acetate) were added anda solution of 0.98 g sodium azide (15.00 mmol, 1.5 eq.) in 5 ml waterwas added drop-wise. The solution was allowed to warm to roomtemperature and stirred for one more hour. Phases were separated, theaqueous phase was extracted two times with EtOAc, combined organicfractions were washed two times with water, dried (MgSO₄) and allvolatiles were removed under reduced pressure.

General Procedure 2 for the Synthesis of O-Ethyl-AlkynylPhosphonamidates from Diethyl Chlorophosphite

A 25-ml Schlenk flask was charged with 173 μl diethyl chlorophosphite(1.20 mmol, 1.2 eq.) under an argon atmosphere, cooled to −78° C. and2.40 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.20 mmol, 1.2eq.) was added drop wise. The yellowish solution was allowed to warm toroom temperature and 1.00 mmol of azide (1.0 eq.) dissolved in 3.0 ml ofTHF or DMF was added and stirred over night at room temperature. 5 ml ofwater were added and stirred for another 2 h. The reaction mixture wasextracted with EtOAc, the combined organic fractions dried (MgSO₄) andsolvents were removed under reduced pressure. The crude product waspurified by flash column chromatography on silica gel or preparativereversed phase HPLC.

4-azidobenzoic acid

The compound was synthesized according to the general procedure 1 from2.00 g 4-aminobenzoic acid (14.58 mmol) and obtained as a yellowishsolid. (2.00 g, 12.26 mmol, 84.1%)

¹H NMR (300 MHz, Chloroform-d) δ=8.11 (d, J=8.4, 2H), 7.11 (d, J=8.4,2H). NMR data was in accordance with literature values (23).

4-azidobenzoic-acid-N-hydroxysuccinimide ester

In a 50-ml round-bottom-flask, 500 mg 4-azidobenzoic acid (3.056 mmol,1.00 eq.), 705 mg N-hydroxysuccinimide (6.112 mmol, 2.00 eq.) and 20 mg4-Dimethylaminopyridine (0.164 mmol, 0.05 eq.) were suspended in 10 mlof dry CH₂C₂. 1.172 g EDC*HCl(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 6.112mmol, 2.00 eq.) were added slowly at 0° C. and the reaction mixture wasallowed to stir at room temperature for two hours. The solvent wasremoved under reduced pressure and the crude product purified by columnchromatography on silicagel (50% EtOAc in hexane) and obtained as whitesolid (763 mg, 2.934 mmol, 96.0%)

¹H NMR (300 MHz, Chloroform-d) δ=8.14 (d, J=8.6, 2H), 7.15 (d, J=8.6,2H), 2.92 (s, 4H). ¹³C NMR (75 MHz, CDCl₃) δ=169.15, 160.97, 146.85,132.42, 121.19, 119.21, 25.59. NMR data was in accordance withliterature values (24).

Ethyl-N-(4-(2,5-dioxo-1-pyrrolidinyl)oxy-carbonyl-phenyl)-P-ethynylphosphonamidate

The compound was synthesized according to the general procedure 2 from173 μl diethyl chlorophosphite (1.20 mmol, 1.20 eq.), 2.40 mlethynylmagnesium bromide solution (0.5 M in THF (tetrahydrofuran), 1.20mmol, 1.20 eq.) and 260 mg 4-azidobenzoic-acid-N-hydroxysuccinimideester (1.00 mmol, 1.00 eq.). The crude phosphonamidate was purified byflash column chromatography on silicagel (100% EtOAc) and obtained as ayellowish solid. (225 mg, 0.643 mmol, 64.3%)

¹H NMR (300 MHz, Chloroform-d) δ=8.05 (d, J=8.6, 2H), 7.37 (d, J=7.4,1H), 7.16 (d, J=8.6, 2H), 4.38-4.13 (m, 2H), 2.96 (d, J=13.2, 1H), 2.90(s, 4H), 1.40 (t, J=7.1, 3H). ¹³C NMR (75 MHz, Chloroform-d) δ=169.59,161.51, 145.64, 132.55, 118.38, 117.59 (d, J=8.0), 88.69 (d, J=50.2),62.93 (d, J=5.2), 25.82, 16.24 (d, J=7.3). ³¹P NMR (122 MHz,Chloroform-d) δ=−10.65. HR-MS for C₁₅H₁₆N₂O₆P⁺ [M+H]⁺ calcd: 351.0740,found 351.0749.

2-(4-Azidophenyl)-ethanol

The compound was synthesized according to the general procedure 1 from1.00 g of 2-(4-Aminophenyl)-ethanol (7.21 mmol) and obtained as brownoil (0.50 g, 3.06 mmol, 42.5%).

¹H NMR (300 MHz, Chloroform-d) δ=7.21 (d, J=8.3, 2H), 6.97 (d, J=8.3,2H), 3.83 (t, J=6.5, 2H), 2.84 (t, J=6.5, 2H), 1.81 (s, 1H). ¹³C NMR (75MHz, CDCl₃) δ=138.26, 135.41, 130.42, 119.18, 63.55, 38.50. NMR data wasin accordance with literature values (25).2-(4-Azidophenyl)-ethyl-4-toluenesulfonate

A 50-ml round-bottom flask was charged with 455 mg of2-(4-Azidophenyl)-ethanol (2.79 mmol, 1.00 eq.), dissolved in 8 mlpyridine and cooled to 0° C. 787 mg of solid tosyl chloride (4.18 mmol,1.50 mmol) was added portion-wise and the mixture was stirred for 4 h atroom temperature, 10 ml of saturate NaCl-solution and 10 ml water wereadded and the yellow solution was extracted three times with EtOAc,combined organic fractions were washed two times with 1N HCl, twice withsaturate NaHCO₃-solution and once with water. The organic layer wasdried (MgSO₄) and all volatiles were removed under reduced pressure.Product was obtained as yellow oil (0.72 g, 2.44 mmol, 87.4%).

¹H NMR (300 MHz, Chloroform-d) δ=7.69 (d, J=8.2, 2H), 7.30 (d, J=8.2,2H), 7.10 (d, J=8.3, 2H), 6.91 (d, J=8.3, 2H), 4.21 (t, J=6.8, 2H), 2.94(t, J=6.8, 2H), 2.45 (s, 3H). ¹³C NMR (75 MHz, CDCl₃) δ=144.66, 138.60,132.96, 132.76, 130.19, 129.69, 127.72, 119.05, 70.34, 34.60, 21.55. NMRdata was in accordance with literature values (26).

2-(4-Azidophenyl)-ethyl phthalimide

A 50-ml round-bottom flask was charged with 4.11 g of2-(4-Azidophenyl)-ethyl-4-toluenesulfonate (12.95 mmol, 1.00 eq.),together with 3.60 g potassium phthalimide (19.42 mmol, 1.50 eq.) anddissolved in 60 ml DMF (N,N-dimethylformamide). The brown solution wasstirred over night at 100° C. All volatiles were removed under reducedpressure, 50 ml of water were added extracted three times with EtOAc,the combined organic fractions were washed two times with water, theorganic layer was dried (MgSO₄) and all volatiles were removed underreduced pressure. The product was used in the next step without furtherpurification. Pure product was obtained by flash column chromatographyon silicagel (10% to 20% EtOAc in n-hexan) as a yellow solid (1.75 g,5.99 mmol, 46.2%). ¹H NMR (600 MHz, Chloroform-d) δ=7.85 (dd, J=5.4,3.1, 2H), 7.73 (dd, J=5.4, 3.1, 2H), 7.25 (d, J=8.4, 2H), 6.96 (d,J=8.4, 2H), 3.93 (dd, J=8.3, 6.8, 2H), 3.00 (dd, J=8.3, 6.8, 2H). ¹³CNMR (151 MHz, CDCl₃) δ=168.12, 138.43, 134.76, 133.96, 132.00, 130.22,123.26, 119.17, 39.14, 33.92.

2-(4-Azidophenyl)-ethylamine hydrochloride

A 100-ml round-bottom flask was charged with 722 mg of2-(4-Azidophenyl)-ethyl phthalimide (2.47 mmol, 1.00 eq.), 144 μlhydrazine hydrate (2.96 mmol, 1.20 eq.), dissolved in 20 ml of dryethanol under argon atmosphere and the solution was refluxed for 4 h.Most of the solvent was removed under reduced pressure, 50 ml water wasadded and the suspension was basified with 1N NaOH. It was extractedthree times with EtOAc, the combined organic fractions were washed twotimes with water, the organic layer was dried (MgSO₄) and all volatileswere removed under reduced pressure. Pure product was obtained by flashcolumn chromatography on silicagel (10% MeOH (methanol) in DCM(dichloromethane)+0.5% N,N-ethyldimethylamine) and lyophilisation fromHCl as yellowish solid (224 mg, 1.14 mmol, 46.2% over two steps). ¹H NMR(600 MHz, Deuterium Oxide) δ=7.29 (d, J=7.6, 2H), 7.05 (d, J=7.6, 2H),3.22 (t, J=7.2, 2H), 2.94 (t, J=7.2, 2H). ¹³C NMR (151 MHz, D₂O)δ=138.81, 133.24, 130.32, 119.40, 40.51, 32.13. NMR data was inaccordance with literature values (27).

Ethyl-N-(4-(2-aminoethyl)phenyl)-P-ethynyl phosphonamidate TFA salt

The compound was synthesized according to the general procedure 2 from181 μl diethyl chlorophosphite (1.26 mmol, 1.20 eq.), 2.52 mlethynylmagnesium bromide solution (0.5 M in THF, 1.26 mmol, 1.20 eq.)and 322 mg 2-(4-azidophenyl)ethyl amine hydrochloride (1.05 mmol, 1.00eq.). The crude phosphonamidate was purified by preparative RP-HPLC(Method C described above) and obtained as brown oil. (209 mg, 0.57mmol, 54.5%)

¹H NMR (300 MHz, Acetonitrile-d₃) δ=7.58 (s, 3H), 7.20-7.01 (m, 4H),6.96 (d, J=8.5, 1H), 4.26-4.05 (m, 2H), 3.42 (d, J=12.8, 1H), 3.08 (d,J=7.8, 2H), 2.88 (dd, J=9.0, 6.4, 2H), 1.31 (t, J=7.1, 3H). ¹³C NMR (75MHz, Acetonitrile-d₃) δ=161.38 (q, J=34.7), 139.20 (d, J=1.3), 131.75,130.66, 119.63 (d, J=7.3), 90.09 (d, J=47.2), 77.02 (d, J=265.0), 63.54(d, J=5.3), 41.92, 33.19, 16.41 (d, J=7.3). ³¹P NMR (122 MHz,Acetonitrile-d₃) δ=−9.71. HR-MS for C12H18N₂O2P⁺ [M+H]⁺ calcd: 253.1100,found 253.1095.

5-((2-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonicacid

The reaction was carried out in DMF. 265 μl of a 100 mM solution ofEthyl-N-(4-(2,5-dioxo-1-pyrrolidinyl)oxy-carbonyl-phenyl)-P-ethynylphosphonamidate (0.0265 mmol, 1.00 eq.) and 1.06 ml of a 50 mM solutionof 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate (0.0530 mmol, 2.00 eq.)together with 795 μl DMF was premixed and 530 μl of a solution of 200 mMDIPEA (0.1060 mmol, 4.00 eq.) was added. The mixture was shaken for 2hours at room-temperature, all volatiles were removed under reducedpressure, the crude mixture was purified by preparative HPLC usingmethod C described above, and the desired compound obtained as a whitesolid after lyophilisation. (9.30 mg, 0.0186 mmol, 70.0%)

¹H NMR (600 MHz, DMSO-d₆) δ=8.78 (d, J=8.5, 1H), 8.57 (t, J=5.7, 1H),8.36 (d, J=8.6, 1H), 8.11 (d, J=8.4, 1H), 7.99 (d, J=7.0, 1H), 7.80 (d,J=8.7, 2H), 7.43 (dd, J=8.5, 7.1, 1H), 7.38 (t, J=8.1, 1H), 7.14 (d,J=8.7, 2H), 6.92 (d, J=7.5, 1H), 4.43 (d, J=12.7, 1H), 4.21-4.05 (m,2H), 3.62 (q, J=6.3, 2H), 3.46 (t, J=6.6, 2H), 1.31 (t, J=7.0, 3H). ¹³CNMR (151 MHz, DMSO-d₆) δ=167.03, 144.64, 143.48, 141.01, 130.59, 128.98,127.65, 126.47, 125.13, 124.62, 123.86, 123.13, 119.62, 117.34 (d,J=7.8), 107.91, 91.69 (d, J=45.5), 77.26 (d, J=260.8), 62.31 (d, J=5.0),45.51, 38.15, 16.42 (d, J=6.9). ³¹P NMR (243 MHz, DMSO) δ=−10.35. HR-MSfor C23H25N₃O6PS⁺ [M+H]⁺ calcd: 502.1196, found 502.1195.

Cy5-O-ethyl-P-alkynyl-phosphonamidate

The Cy5-COOH was synthesized according to a procedure, previouslypublished by our lab (28). A 5-ml-round bottom flask was charged with33.2 mg Cy5-COOH (0.0628 mmol, 1.00 eq.), 35.8 mg HATU((1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate), 0.0942 mmol, 1.5 eq.) and 200 μl DMF. Thedeep blue solution was cooled to 0° C. and 32 μl DIPEA(N,N-diisopropylethylamine, 0.1884 mmol, 3.0 eq.) were added. After 5minutes a solution of 23 mg Ethyl-N-(4-(2-aminoethyl)phenyl)-P-ethynylphosphonamidate TFA salt (0.0628 mmol, 1.00 eq.) in 300 μl DMF wereadded drop-wise. The solution was allowed to warm to room-temperatureand stirred for 2 hours. All volatiles were removed under reducedpressure and the crude product was purified by flash columnchromatography on silicagel (0% to 5% MeOH in DCM) and obtained as bluesolid. (45 mg, 0.0590 mmol, 93.9%).

¹H NMR (600 MHz, Chloroform-d) δ=7.88 (td, J=13.0, 4.9, 2H), 7.43-7.33(m, 4H), 7.23 (t, J=7.4, 2H), 7.15-7.07 (m, 4H), 7.01 (d, J=8.4, 2H),6.72 (t, J=12.5, 1H), 6.46 (bs, 1H), 6.18 (dd, J=13.6, 8.5, 2H), 6.11(q, J=7.6, 1H), 4.27-4.09 (m, 2H), 3.98 (t, J=7.6, 2H), 3.56 (s, 3H),3.43 (q, J=6.9, 2H), 2.97 (d, J=12.8, 1H), 2.75 (t, J=7.5, 2H), 2.25 (t,J=7.3, 2H), 1.81 (p, J=8.0, 2H), 1.73-1.67 (m, 2H), 1.70 (s, 6H), 1.69(s, 6H), 1.55-1.42 (m, 2H), 1.35 (t, J=7.1, 3H). ¹³C NMR (151 MHz,CDCl3) δ=173.64, 173.19, 173.11, 153.34, 152.99, 142.72, 141.90, 141.17,140.89, 136.88, 133.32, 129.69, 128.78, 128.66, 126.32, 126.22, 125.34,125.15, 122.21, 122.13, 118.60, 118.53, 110.83, 110.36, 103.77, 103.64,88.54, 88.23, 75.27, 62.46, 49.40, 49.17, 44.22, 41.03, 35.94, 34.78,27.96, 27.90, 27.84, 27.09, 26.32, 25.24, 16.17, 16.11, 16.04. ³¹P NMR(243 MHz, CDCl3) δ=−9.08.

Staudinger-Induced Thiol Addition with Alkynyl-Phosphonites for theGeneration of Antibody Drug Conjugates (ADCs)

As set our herein above, we were able to show that a modification offull length igG antibodies with alkyne- and alkene-phosphonamidates ispossible. In the above examples we used Cetuximab as a model antibodyand modified the interchain-disulfides with a biotinylatedphorsphonamidate via the reduction and alkylation protocol, previouslydescribed by Senter and coworkers (29). This concept was furtherdeveloped towards a feasible system for the generation of ADCs byphosphonamidate mediated conjugation the highly potent tubulin bindingcytotoxin MMAE and the Her2 binding antibody Trastuzumab.

Similar to our above studies with Cetuximab, we reduced the inter-chaindisulfide bonds of Trastuzumab with dithiothreitol (DTT) and carried outCys-conjugation reactions with different electrophilic biotinderivatives, including maleimide, iodoacetamide andalkyne-phopshonamidate (phosphonamidate-labelling), to have a directcomparison to state-of-the art techniques. The latter was synthesized bythe Staudinger phosphonite reaction protocol in 72% overall yield. Theantibody-labelling reactions were carried out with and without priorreduction of the disulfide bonds to probe the chemoselectivity of theCys-conjugation reactions (FIGS. 16A-16B). Western blot analysisrevealed sufficient labelling for all of the tested biotin derivativeswith reduced trastuzumab. Most strikingly, we observed high reactivityof maleimides with non-reduced trastuzumab, which was further confirmedby trypsin digestion and MS/MS analysis. In contrast,phosphonamidate-labelling demonstrated outstanding selectivity forCys-residues (FIGS. 16A-16B).

FIGS. 16A-16B shows: FIG. 16A: Trastuzumab modification with threedifferent Cys-reactive biotin derivatives. Disulfide reduction wascarried out with 1000 eq. DTT in 50 mM borate containing PBS for 30minutes at 37° C. Excess DTT was removed by size exclusionchromatography. Labelling was conducted with 35 eq. biotin derivativewith a final DMSO content of 1% in a Buffer containing 50 mM NH₄HCO₃ and1 mM EDTA, pH 8.5 for the amidate and PBS containing 1 mM EDTA, pH 7.4for the other two compounds. FIG. 16B: Western blot analysis. Lane 1 and5: untreated antibody. Lane 2-4: reactions with prior DTT treatment.Lane 6-8: Control reactions without prior DTT treatment.

Phosphonamidate-linked ADCs were generated from the very efficientantimitotic toxin MMAF and the FDA approved Her2-addressingantiproliferative antibody trastuzumab (FIGS. 17A-17C). To investigaterelease of the toxic payload, ADCs with a cathepsin B cleavage side(Valine-Citruline linker VC) were prepared between the antibody and thetoxin. Amidate-VC-PAB-MMAF constructs were synthesized based on apreviously described procedure, as depicted in Scheme 27.

Conjugation to Trastuzumab was carried out in 50 mM ammoniumbicarbonatebuffer at pH 8.5 for 16 hours at 14° C., after reduction of theinterchain-disulfide bonds with DTT and removal of the excess reducingagent by Zeba™ Spin desalting columns.

FIGS. 17A-17C show: Trastuzumab modification with phosphonamidatemodified, cathepsin cleavable MMAF (Amidate-VC-PAB-MMAF). FIG. 17A:Reaction scheme reduction and alkylation of interchain disulfides. FIG.17B: SDS-PAGE analysis of the reaction. FIG. 17C. Deconvolutet MSspectra of the antibody fragments after deglaycosylation with PNGase Fand reduction with DTT. LC: Light chain; HC: Heavy chain; mod:Amidate-VC-PAB-MMAF.

An average loading of 4.6 drug molecules per antibody was determined byESI-MS after deglycosylation and reduction. We approximated thedrug-to-antibody ratio (DAR) with the mass signal intensities of theheavy- and light-chain species bearing different degrees ofmodification.

The obtained Phosphonamidate-ADC conjugates were evaluated in apreviously established Her2 based proliferation assay with two differentHer2-overexpressing cell lines BT474 and SKBR3 (30). The Her2-nonoverexpressing cell line MDAMB468 was used as a control to proof Her2selectivity. Phosphonamidate-linked conjugates were compared to amaleimide-linked cathepsin B-cleavable trastumzumab MMAF conjugate.These experiments clearly demonstrate that phosphonamidate-labelledMMAF-ADCs enable sufficient and selective killing of Her2 overexpressingcells. The measured IC₅₀-values were at least as good as the comparedmaleimide controls (FIG. 18 ). It is important to note, that it is notto be expected that the advantages of phosphonamidate-labelling have apositive effect on in vitro cell killing efficiency when compared tomaleimide chemistry.

FIG. 18 shows: Increased antiproliferative potency of MMAF linkedtrastuzumab on two different Her2 overexpressing cell lines (BT474 andSKBR3) and one control (MDAMB468). Plots depict the number ofproliferating cells after 4 days of antibody treatment in dependency ofthe antibody concentration. Trastuzumab alone (pink),trastuzumab-phosphonamidate-MMAF (blue) and trastuzumab-maleimide-MMAF(green).

Procedures for the Staudinger-Induced Thiol Addition withAlkynyl-Phosphonites for the Generation of Antibody Drub Conjugates(ADCs) N-(4-azidobenzoyl)-L-valine

A 50-ml Schlenk-flask was charged with 1.00 g of 4-azidobenzoic acid(6.13 mmol, 1.00 eq.) and suspended in 8.5 ml of dry DCM(dichloromethane) together with a drop of DMF (N,N-dimethylformamide)under argon. 630 μl of oxalylchloride were added drop-wise at 0° C. andthe reaction mixture was stirred at room temperature for 2 h until thesolution became clear. All volatiles were removed under reduced pressureand the corresponding solid was redissolved in 4 ml of DMF. Thecorresponding solution was added drop-wise at 0° C. to a solution of 720mg L-valin (6.13 mmol, 1.00 eq.) and 612 mg sodium hydroxide (15.33mmol, 2.50 eq.) in 8 ml water and stirred for 2 more hours. The solutionwas acidified with 1 N HCl and extracted three times with diethylether.The organic fractions were pooled, dried (MgSO₄) and the solvents wereremoved under reduced pressure. Pure product was obtained by flashcolumn chromatography on silicagel (30% EtOAc, 0.5% formic acid inn-hexane) as colourless fume. (954 mg, 4.96 mmol, 80.9%)

¹H NMR (600 MHz, Chloroform-d) δ=10.12 (s, 1H), 7.79 (d, J=8.6, 2H),7.05 (d, J=8.6, 2H), 6.79 (d, J=8.5, 1H), 4.76 (dd, J=8.5, 4.9, 1H),2.33 (pd, J=6.9, 4.9, 1H), 1.03 (d, J=6.9, 3H), 1.01 (d, J=6.9, 3H). ¹³CNMR (151 MHz, CDCl₃) δ=175.82, 167.28, 144.03, 130.17, 129.13, 119.20,77.16, 57.79, 31.40, 19.16, 17.99. HR-MS for C12H15N₄O3⁺ [M+H]⁺ calcd:263.1139, found 263.1151.

N-(4-azidobenzoyl)-L-valine-anhydride

In a 100-ml round-bottom flask, 954 mg N-(4-azidobenzoyl)-L-valine (3.64mmol, 1.00 eq.), 750 mg dicyclohexylcarbodiimide (3.64 mmol, 1.00 eq.),418 mg N-hydroxysuccinimide (3.64 mmol, 1.00 eq.) and 9 mg4-(dimethylamino)-pyridine (0.07 mmol, 0.02 eq.) were dissolved in 25 mlof THF and stirred over night at room temperature. The reaction mixturewas filtered, the solids were washed several times with THF, the solventwas removed under reduced pressure and the crude product was purified byflash column chromatography on silicagel (20 to 40% EtOAc in n-hexane).The compound was isolated as white powder (513 mg, 1.01 mmol, 55.7%)

¹H NMR (600 MHz, Chloroform-d) δ=8.01 (d, J=8.7, 2H), 7.13 (d, J=8.7,2H), 4.29 (d, J=4.6, 1H), 2.39 (heptd, J=6.9, 4.6, 1H), 1.16 (d, J=6.9,3H), 1.03 (d, J=6.9, 3H). ¹³C NMR (151 MHz, CDCl₃) δ=177.52, 160.90,144.51, 129.60, 122.43, 119.30, 70.68, 31.28, 18.76, 17.57.

N-(4-azidobenzoyl)-L-valine-L-citrulline

In a 50-ml round-bottom flask, 380 mgN-(4-azidobenzoyl)-L-valine-anhydride (0.75 mmol, 1.00 eq.) weredissolved in 2 ml of 1,2-Dimethoxyethane and cooled to 0° C. A solutionof 351 mg L-citrulline (1.50 mmol, 2.00 eq.) and 144 mg sodiumhydrogencarbonate (2.25 mmol, 3.00 eq.) in 4 ml H₂O and 2 ml THF(tetrahydrofuran) was added dropwise and stirred over night at roomtemperature. All volatiles were removed under reduced pressure and thecrude product was purified by flash column chromatography on silicagel(10% MeOH, 0.5% formic acid in CH₂Cl₂). The compound was isolated ascolourless oil (312 mg, 0.74 mmol, 99.0%).

¹H NMR (600 MHz, DMSO-d₆) δ=8.31 (d, J=8.8, 1H), 8.27-8.21 (m, 1H), 7.96(d, J=8.6, 2H), 7.20 (d, J=8.6, 2H), 6.05 (t, J=5.5, 1H), 5.47 (s, 2H),4.37 (t, J=8.3, 1H), 4.18 (td, J=8.1, 5.1, 1H), 2.98 (q, J=6.4, 2H),2.15 (dq, J=13.6, 6.8, 1H), 1.78-1.68 (m, 1H), 1.68-1.56 (m, 1H),1.51-1.35 (m, 2H), 0.96 (d, J=6.8, 3H), 0.94 (d, J=6.8, 3H). ¹³C NMR(151 MHz, DMSO) δ=174.09, 171.54, 165.99, 159.40, 142.77, 131.36,129.93, 119.23, 59.31, 52.57, 49.07, 30.77, 29.01, 27.07, 19.75, 19.28.HR-MS for C₁₈H26N₇O5⁺ [M+H]⁺ calcd: 420.1990, found 420.1990.

N-(4-azidobenzoyl)-L-valine-L-citrulline-4-aminobenzyl alcohol

In a 50-ml round-bottom flask, 330 mgN-(4-azidobenzoyl)-L-vaine-L-citrulline (0.787 mmol, 1.0 eq.) and 107 mg4-aminobenzyl alcohol (0.866 mmol, 1.10 eq.) were dissolved in 8 mlCH₂Cl₂ and 4 ml MeOH (methanol) under an argon atmosphere and cooled to0° C. 390 mg N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.574 mmol,2.00 eq.) were added portion-wise and the resulting solution was allowedto warm to room temperature overnight. All volatiles were removed underreduced pressure and the crude product was isolated by flash columnchromatography on silicagel (10% to 15% MeOH in CH₂Cl₂) and obtained aswhite solid (164 mg, 0.313 mmol, 39.8%). Enantiomeric pure compound wasisolated by preparative HPLC (Method D described above under “Proceduresfor the introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”) and obtained as a white solid afterlyophilisation.

¹H NMR (600 MHz, DMSO-d₆) δ=9.93 (s, 1H), 8.32 (d, J=8.4, 1H), 8.21 (d,J=7.6, 1H), 7.96 (d, J=8.6, 2H), 7.55 (d, J=8.6, 2H), 7.24 (d, J=8.6,2H), 7.21 (d, J=8.6, 2H), 6.12 (bs, 2H), 4.44 (s, 2H), 4.46-4.40 (m,1H), 4.36 (t, J=8.1, 1H), 3.09-2.93 (m, 2H), 2.24-2.04 (m, J=6.7, 1H),1.84-1.58 (m, 2H), 1.55-1.34 (m, 2H), 0.95 (d, J=6.7, 3H), 0.94 (d,J=6.7, 3H). ¹³C NMR (151 MHz, DMSO) δ=171.62, 170.79, 166.15, 159.46,142.83, 137.95, 137.91, 131.29, 129.96, 127.38, 119.34, 119.26, 63.07,59.56, 53.64, 39.20, 30.61, 29.88, 27.16, 19.79, 19.37. HR-MS forC₂₅H₃₃NO₅ ⁺ [M+H]⁺ calcd: 525.2568, found 525.2563. [α]_(D) ²⁴=−49.6(c=0.81; MeOH)

N-(4-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzylalcohol

The compound was synthesized according to the general procedure from 230μl diethyl chlorophosphite (0.925 mmol, 5.0 eq.), 1.85 mlethynylmagnesium bromide solution (0.5 M in THF, 0.925 mmol, 5.0 eq.)and 97 mg N-(4-azidobenzoyl)-L-valine-L-citrulline-4-aminobenzyl alcohol(0.185 mmol, 1.0 eq.). The crude phosphonamidate was purified bypreparative HPLC (method C described above under “Procedures for theintroduction of the alkyne-phosphonamidate moiety by generic buildingblocks via an amide bond”) and obtained as a white solid afterlyophilisation. (60 mg, 0.098 mmol, 52.9%).

¹H NMR (600 MHz, DMSO-d₆) δ=9.96 (s, 1H), 8.80 (d, J=8.5, 1H), 8.22 (d,J=7.7, 1H), 8.11 (dd, J=8.6, 1.9, 1H), 7.82 (d, J=8.7, 2H), 7.56 (d,J=8.4, 2H), 7.24 (d, J=8.4, 2H), 7.14 (d, J=8.7, 2H), 4.39-4.46 (m, 4H),4.33 (t, J=8.1, 1H), 4.20-4.03 (m, 2H), 3.02 (ddt, J=38.3, 13.4, 6.8,2H), 2.20-2.09 (m, J=6.9, 1H), 1.79-1.57 (m, 2H), 1.53-1.35 (m, 2H),1.31 (t, J=7.1, 3H), 0.95 (d, J=6.9, 3H), 0.93 (d, J=6.9, 3H). ¹³C NMR(151 MHz, DMSO-d₆) 5=171.74, 170.81, 166.59, 159.53, 143.51, 137.93 (d,J=9.0), 129.31, 127.57, 127.38, 119.34, 117.24 (d, J=7.7), 91.68 (d,J=45.5), 77.25 (d, J=261.1), 63.06, 62.29 (d, J=5.0), 59.45, 53.61,39.27, 30.70, 29.82, 27.03, 19.82, 19.32, 16.42 (d, J=6.9). ³¹P NMR (243MHz, DMSO-d₆) δ=−13.28, −13.32. HR-MS for C₂₉H₄₀N₆O₇P⁺ [M+H]⁺ calcd:615.2691, found 615.2716.

N-(4-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzyl-4-nitrophenylcarbonate

A 5-ml round-bottom flask was charged with 31 mgN-(4-(0-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzylalcohol (0.050 mmol, 1.00 eq.) and 31 mg Bis(4-nitrophenyl) carbonate(0.101 mmol, 2.00 eq.). The solids were dissolved in 140 μl of DMF(N,N-dimethylformamide) and 17.4 μl DIPEA (N,N-diisopropylethylamine,0.101 mmol, 2.00 eq.) were added. The yellow solution was stirred for 1h at room temperature and the solution was added to 30 ml of ice-colddiethyl ether. The precipitate was collected by centrifugation,redissolved in DMF and again precipitated with ether. The procedure wasconducted three times in total and finally the solid was dried underhigh vacuum conditions. The compound was isolated in quantitative yieldsand sufficiently pure for the next step. Analytical pure material waspurified by preparative HPLC using method C described above under“Procedures for the introduction of the alkyne-phosphonamidate moiety bygeneric building blocks via an amide bond”.

¹H NMR (600 MHz, DMSO-d₆) δ=10.10 (s, 1H), 8.79 (d, J=8.5, 1H), 8.32 (d,J=9.1, 1H), 8.23 (d, J=7.4, 1H), 8.07 (dd, J=8.5, 2.2, 1H), 7.81 (d,J=8.7, 2H), 7.66 (d, J=8.5, 2H), 7.57 (d, J=9.1, 1H), 7.42 (d, J=8.5,2H), 7.13 (d, J=8.7, 2H), 5.25 (s, 2H), 4.47-4.40 (m, 2H), 4.34 (t,J=8.0, 1H), 4.20-4.05 (m, 2H), 3.01 (ddt, J=47.1, 13.4, 6.8, 2H),2.20-2.09 (m, J=6.8, 1H), 1.80-1.59 (m, 2H), 1.55-1.35 (m, 2H), 1.30 (t,J=7.0, 3H), 0.95 (d, J=6.7, 3H), 0.93 (d, J=6.7, 3H). ¹³C NMR (151 MHz,DMSO-d₆) δ=171.79, 171.17, 166.58, 159.44, 155.75, 152.42, 145.63,143.50, 139.83, 129.95, 129.77, 129.30, 127.59, 125.86, 123.08, 119.51,117.24 (d, J=7.8), 91.67 (d, J=45.6), 77.26 (d, J=261.0), 70.71, 62.26(d, J=5.0), 59.31, 53.68, 39.14, 30.71, 29.76, 27.19, 19.80, 19.30,16.41 (d, J=6.9). ³¹P NMR (243 MHz, DMSO) δ=−10.39, −10.44. HR-MS forC₈₆H₄₃N₇O₁₁P⁺ [M+H]⁺ calcd: 780.2753, found 780.2744.

Amidate-Val-Cit-Pab-MMAF

A 15-mL falcon-tube was charged with 14.35 mgN-(4-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzyl-4-nitrophenylcarbonate (0.0184 mmol, 1.00 eq.), 0.50 mg 1-Hydroxybenzotriazole(0.0037 mmol, 0.20 eq.) and 13.15 mg MMAF (monomethylauristatin F,0.0184 mmol, 1.00 eq.). The solids were dissolved in 250 ml dry DMF and25 ml pyridine and heated to 60° C. over-night. All volatiles wereremoved under reduced pressure, the crude product was purified bysemi-preparative HPLC using method E described above under “Proceduresfor the introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”, and the desired compound obtained asa white solid after lyophilisation. (4.84 mg, 0.0035 mmol, 19.2%). HR-MSfor C₆₉H₁₀₄N₁₁O₁₆P²⁺ [M+2H]²⁺ calcd: 686.8695, found 686.8694.

FIG. 19 shows the UPLC-UV purity of phosphonamidate-Val-Cit-Pab-MMAF.

Trastuzumab Production

Trastuzumab expression and purification was executed as previouslypublished with an additional final purification by gel filtration on aSuperdex 200 Increase 10/300 from GE with phosphate-buffered saline(PBS) and flow rate of 0.75 ml/min (30).

General Procedure for the Modification of Trastuzumab Via theReduction/Alkylation Protocol

The general procedure for the modification of Trastuzumab via thereduction/alkylation protocol is shown in FIG. 44 .

Trastuzumab modification was carried out by incubating freshly expressedantibody (c=0.55 mg/ml) in a buffer containing 50 mM sodium borate and 4mM DTT in PBS (pH 8.0) with a total volume of 80 μl at 37° C. for 40min. Excess DTT removal and buffer exchange to a solution containing 50mM NH₄HCO₃ and 1 mM EDTA (pH 8.5) was conducted afterwards using 0.5 mLZeba™ Spin Desalting Columns with 7K MWCO (Thermo Fisher Scientific,Waltham, United States). 1.60 μl of a solution containing 13 mM amidatein DMSO was added quickly. And the mixture was shaken at 800 rpm and 14°C. for 16 hours. Excess amidate was again removed by buffer exchange tosterile PBS using 0.5 mL Zeba™ Spin Desalting Columns with 7K MWCO.

Cell Based Antiproliferation Assays

Antiproliferation assays were conducted as previously reported (30) withthe following minor changes:

-   -   For MDAMB468 cells, a reduced amount of 2*10³ cells were seeded        in each well of a 96-well optical cell culture plate        supplemented with 100 μL culture media.    -   Images were acquired with an Operetta High-Content Imaging        system (PerkinElmer, Waltham, Mass., USA) equipped with a 20×        high NA objective.    -   Cell counts were calculated from duplicates

Staudinger-Induced Thiol Addition with Alkynyl-Phosphonites for theGeneration of Antibody Fluorophore Conjugates (AFCs)

In a similar manner, as described above under “Staudinger-induced thioladdition with alkynyl-phosphonites for the generation of Antibody DrugConjugates (ADCs)” the fluorescent dye Cy5 was conjugated to Trastuzumabto generate an antibody-fluorophore conjugate. Synthesis ofCy5-O-ethyl-P-alkynyl-phosphonamidate was conducted as described aboveunder “Introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”. The obtained Phosphonamidate-AFCconjugates were evaluated by immunostaining of two differentHer2-overexpressing cell lines BT474 and SKBR3. The Her2-nonoverexpressing cell line MDAMB468 was used as a control to proof Her2selectivity. Sufficient membrane staining after cell fixation wasobserved the two Her2-expressing cell lines, while the Her2-nonexpressing cell lines did not show increased fluorescence.

FIG. 20 : Depicted are immunostainings of fixed cells over expressingthe cell surface receptor Her2 (BT474 and SKBR3) or exhibiting low Her2expression levels (MDAMB468). The AFC Trastuzumab-Amidate-Cy5 showsclear localization to the plasma membrane for Her2+ cell lines and nostaining of Her2− cells. The merged images show the DAPI signal in blueand the Tras-phosphonamidate-Cy5 signal in red. Scale bar represents 10μm.

Procedures for the Staudinger-Induced Thiol Addition withAlkynyl-Phosphonites for the Generation of Antibody FluorophoreConjugates (AFCs)

The general procedures for the Staudinger-induced thiol addition withalkynyl-phosphonites for the generation of Antibody FluorophoreConjugates (AFCs) are shown in FIG. 45 .

Trastuzumab-Cy5 conjugates were synthesized according to the generalprocedure, described above under “Staudinger-induced thiol addition withalkynyl-phosphonites for the generation of Antibody Drug Conjugates(ADCs) with the following slight modifications: the amidate equivalentswere raised to 130, and the DMSO (dimethylsulfoxide) content was raisedto 5% (more precisely, from 2% to 5%) to solubilize the Cy5.

AFC Imaging Procedure

BT474, SKBR3 and MDAMB468 were seeded on sterile cover slips andincubated ON at 37° C., 5% CO2 for cell attachment. Cells were washedthree times with 1×PBS prior to fixation for 10 min in 1×PBS/4% PFA(formaldehyde). Fixation was stopped by the addition of an equal volume1×PBST (PBS+0.05% Tween20) followed by two more washes with PBST. AFCswere added to a final concentration of 5 μg/mL and incubated for 1 h atRT. Unbound AFC was removed by three washes with PBS.

Images were acquired on a Leica SP5 confocal microscopy system equippedwith a 63×1.40 Oil immersion objective. Laserlines 405 nm and 594 nmwere used in combination with standard DAPI and Cy5 filter settings.Image processing was carried out with ImageJ 1.5.1 h software extendedby the Fiji processing package.

Stability Studies of the Phosphonamidate Linkage

To study the stability of the phosphonamidate bond in complex systems ascell lysate or serum, a dye-quencher pair was synthesized whichgenerates a fluorescent signal upon cleavage of the phosphonamidatebond. Conjugates consist of the fluorescent dye EDANS, the quencherDABCYL and an attached peptide to ensure water solubility of theconjugates (FIGS. 21A-21E). A maleimide linked conjugate was synthesizedfor comparison experiments (FIG. 21B).

FIGS. 21A-21E show: FIG. 21A: Structure of the phosphonamidate linkedFRET conjugate. FIG. 21B: Structure of the maleimide linked FRETconjugate. FIG. 21C: Principle of the fluorescence-quencher basedreadout. Conjugates were incubated at room temperature at aconcentration of 10 μM. Measurements were performed at least intriplicates in a 96-well plate. FIG. 21D: Fluorescence increase wasmonitored over time. HCl samples were neutralized before themeasurements. Lysate was freshly prepared from HeLa-cells, lysed in PBS.Serum originated from human blood. FIG. 21E: Comparison of aphosphonamidate- and a maleimide-linked dye-quencher pair duringexposure to 1000 eq. glutathion in PBS.

As shown in FIGS. 21A-21E, the phosphonamidate adducts show a highstability in PBS, HeLA cell lysate and human serum, whereas only strongacidic conditions (1N HCl) lead to phosphonamidate bond cleavage. TheFRET-conjugates were also exposed to a high excess of glutathione. After2 days of incubation with 1000 eq. of glutathione at physiological pH,15% of the maleimide linkage was cleaved, while 99% of thephosphonamidate adducts were still intact (FIG. 21C).

In the next experiment, we probed whether the modification element ofphosphonamidate-labelled or maleimide-labelled ADCs is transferred toserum proteins in the presence of thiols, as the stability of ADCs forseveral days is crucial during circulation in the blood stream.Trastuzumab modified with different biotin derivatives (FIGS. 22A-22B)was exposed to serum-like albumin concentrations of 0.5 mM, incubated at37° C., and transfer of the modification from the antibody to the serumprotein was monitored by western blotting (FIG. 22B). A significanttransfer of the biotin to BSA (bovine serum albumin) at serumconcentrations was observed for maleimide linkage while thephosphonamidate-linkage was stable under the tested conditions. Takentogether, these stability experiments experiment clearly point tosuperior stability of the phosphonamidate-labelled ADCs as compared tomaleimide-labelled ADCs potentially leading to a reduction of off-targettoxicity when compared to conventional maleimide-linked conjugates.

FIGS. 22A-22B show: Transfer of the antibody modification to serumproteins. FIG. 22A: Tratuzumab-biotin conjugates were incubated at aconcentration of 3 μM with 500 μM BSA in PBS at 37° C. FIG. 22B: Biotintransfer to albumin was monitored by western blot analysis. Lane 1:Untreated maleimide conjugate. Lane 2-5: Analysis of the BSA exposedmaleimide adduct after 0, 1, 2 and 5 days. Lane 6: Untreated amidateconjugate. Lane 6-10: Analysis of the BSA exposed amidate adduct after0, 1, 2 and 5 days.

Procedures for the Stability Studies of the Phosphonamidate LinkageDABCYI-Cys Peptide

DABCYI-Cys peptide was synthesized by standard Fmoc-based chemistry in alinear synthesis by manual coupling. 0.1 mmol of Rink amide resin(subst: 0.4 mmol/g) was added to a reaction vessel and synthesis wasperformed with five-fold amino acid excess. Fmoc de-blocking wasachieved by resin treatment with 20% piperidine in DMF twice for 5minutes. Coupling was achieved by addition of HOBt/HBTU/DIPEA (5 eq./5eq./10 eq) in DMF for 45 min. After the final Cys coupling, 5 eq. of theDABCYL acid was coupled with 5 eq. HATU and 10 eq. DIPEA in DMF for 45min. The peptide was cleaved of the resin by addition of TFA/DTT/TIS(95/2.5/2.5, w,w,w) within 3 h. Subsequently, the peptide wasprecipitated by the addition of ice-cold diethyl ether. The precipitatewas collected by centrifugation, dried and purified by preparative HPLC(method C described above under “Procedures for the introduction of thealkyne-phosphonamidate moiety by generic building blocks via an amidebond”). The peptide was obtained as a red solid in a yield of 35.8%(38.2 mg, 35.8 μmol). ESI-MS for C48H66N₁₂O14S⁺ [M+2H]⁺ calcd: 533.23,found 533.34.

DABCYI-Cys Peptide Phosphonamidate EDANS Adduct

A 1.5-ml Eppendorf tube was charged with 263 μl of a solution ofDABCYI-Cys peptide (20 mM) in 50 mM NH₄HCO₃ at a pH of 8.5. 158 μl 50 mMNH₄HCO₃ at a pH of 8.5 and 105 μl of a solution of EDANS amidate (100mM) in DMF was added to give a final concentration of 20 mM peptide and10 mM amidate in 20% DMF/Buffer. The tube was shaken at 800 rpm at roomtemperature for 3 h. All volatiles were removed under reduced pressureand the crude product purified by semi-preparative HPLC (method Edescribed above under “Procedures for the introduction of thealkyne-phosphonamidate moiety by generic building blocks via an amidebond”). The peptide was obtained as a red solid ESI-MS forC₇₁H₉₀N₁₅O₂₀PS₂ ⁺ [M+2H]⁺ calcd: 783.78, found 784.47.

DABCYI-Cys Peptide Maleimide EDANS Adduct

A 1.5-ml Eppendorf tube was charged with 188 μl of a solution ofDABCYI-Cys peptide (20 mM) in PBS. 188 μl of a solution of EDANSmaleimide (40 mM) in DMF was added to give a final concentration of 10mM peptide and 20 mM maleimide in 50% DMF/Buffer. The tube was shaken at800 rpm at room temperature for 3 h. All volatiles were removed underreduced pressure and the crude product purified by semi-preparative HPLC(method E described above under “Procedures for the introduction of thealkyne-phosphonamidate moiety by generic building blocks via an amidebond”). The peptide was obtained as a red solid. ESI-MS forC₆₆H₈₃N₁₅O₂₀S₂ ⁺ [M+2H]⁺ calcd 734.77, found. 734.79

Stability Studies of the Dabcyl-EDANS Adducts

Stabilities studies were conducted in 96-well plate (Corning 3615, blackwith clear, flat bottom) at least in triplicates. 5 μl of a 200 μM Stocksolution of the Dabcyl-EDANS adducts and 95 μl of the respective testsolutions were added to each well.

HeLa cell lysate was generated from approximately 1*10⁷ cells, lysed in400 μl PBS by sonification. Cells were grown on a 75 cm² cell cultureplate, washed twice with PBS and harvested with a cell scraper. Humanserum was purchased from Sigma Aldrich. Glutathione was dissolved at aconcentration of 10 mM in PBS and the pH was adjusted to 7.4. 1N HClstudies were conducted at 200 μM, neutralized to pH 7 and diluted to 10μM before fluorescence measurements.

Fluorescence was measured on a Tecan Safire plate reader. Excitation:336 nm, emission: 490 nm, bandwidth: 5 nm at 20° C.

Incubation of Trastuzumab-Biotin Conjugates with BSA

Trastuzumab-Biotin conjugates were incubated at a concentration of 3 μMin PBS with a final concentration of 0.5 mM BSA at 37° C. Samples weredrawn after 0, 1, 2 and 5 days, deep frozen in liquid Nitrogen andfinally subjected to SDS/Page and western blot analysis.

Further Kinetic Investigations of the Thiol Addition

To study the kinetics of the thiol addition to alkyne-phosphonamidatesat low concentrations, a fluorescent EDANS-based phosphonamidate wassynthesized as described in chapter 1.1. Addition of glutathione as amodel substrate was probed over time by fluorescence HPLC. Peakintegration and normalization to unconjugated EDANS as an internalstandard was applied to determine the second order rate constant of thereaction. A second order rate constant of 37.32±0.41 I/mol*s wasmeasured.

FIGS. 23A-D show: Determination of the second order rate constant of thethiol addition. FIG. 23A: Reaction of the EDANS phosphonamidate withglutathione. FIG. 23B: Fluorescence HPLC trace after 30 min reactiontime. FIG. 23C: Monitoring of the phosphonamidate decrease over time.FIG. 23D: Plot of the inverse concentration against reaction time. Errorbars represent the mean of three replicates (n=3).

Procedures for the Further Kinetic Investigations of the Thiol Addition

Glutathione addition to the EDANS-phosphonamidate was conducted at afinal concentration of 0.1 mM amidate, 0.1 mM glutathione and 0.02 mMEDANS as an internal standard in 50 mM NH₄HCO₃-buffer containing 1 mMEDTA at pH 8.5 with 1% DMF. 2.5 μl of a 20 mM stock solution ofEDANS-phosphonamidate in DMF was premixed with 488 μl buffer and 5 μl ofa 2 mM stock solution of EDANS in a 1:1 mixture of DMF and buffer. Thereaction was started by the addition of 5 μl of a 10 mM solution ofglutathione in buffer. 10 μl samples were drawn at 0, 15, 30, 60, 120,240 and 480 minutes and acidified with 190 μl 10 mM NaOAc-Buffer (pH5.0) and subjected to fluorescence HPLC analysis.

Synthesis of Further Phosphonites

Further, the O-substituent of the alkyne phosphonites was varied asshown in Scheme E2, and electron-rich phosphonites E1 to E5 weresynthesized:

Procedures for the Synthesis of Further Phosphonites E1 to E5 GeneralProcedure for the Synthesis of O-Substituted Alkynyl Phosphonamidatesfrom Bis(Diisopropylamino)Chlorophosphine

A 25-ml Schlenk flask was charged with 267 mgbis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.) under anargon atmosphere, cooled to 0° C. and 2.20 ml ethynylmagnesium bromidesolution (0.5 M in THF, 1.10 mmol, 1.10 eq.) was added drop wise. Theyellowish solution was allowed to warm to room temperature and stirredfor further 30 minutes. The respective alcohol, dissolved in 5.56 ml1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol) was added and thewhite suspension was stirred over night at room temperature. Thereaction mixture was directly placed on a silica gel flash column.

Di-(2-(2-Hydroxyethoxy)Ethyl) Ethynylphosphonite (Compound E1)

The compound was synthesized according to the above “General procedurefor the synthesis of O-substituted alkynyl phosphonamidates frombis(diisopropylamino)chlorophosphine” from 267 mgbis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 mlethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.),1.06 g 2-(2-Hydroxyethoxy)ethan-1-ol (10.00 mmol, 10.00 eq.), 5.56 ml1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol) and purified by flashcolumn chromatography on silicagel (5% MeOH in CH₂Cl₂). The compound wasobtained as a yellowish oil. (112 mg, 0.421 mmol, 42.1%).

¹H NMR (300 MHz, Chloroform-d) δ=4.14-3.98 (m, 4H), 3.65-3.59 (m, 4H),3.58-3.49 (m, 8H), 3.15 (d, J=2.4, 1H). ¹³C NMR (75 MHz, Chloroform-d)δ=92.52, 92.50, 84.61, 83.98, 72.60, 70.72 (d, J=4.0), 67.20 (d, J=6.0),61.44. ¹³C NMR (75 MHz, Chloroform-d) δ=92.51 (d, J=1.4), 84.30 (d,J=46.8), 72.60, 70.72 (d, J=4.0), 67.20 (d, J=6.0), 61.44. ³¹P NMR (122MHz, CDCl₃) δ=131.97.

Di-(3-Butinyl) Ethynylphosphonite (Compound E2)

The compound was synthesized according to the above “General procedurefor the synthesis of O-substituted alkynyl phosphonamidates frombis(diisopropylamino)chlorophosphine” from 267 mgbis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 mlethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.),189 μl 3-Butyn-1-ol (2.50 mmol, 2.50 eq.), 5.56 ml 1H-tetrazole solution(0.45 M in MeCN, 2.50 mmol) and purified by flash column chromatographyon silicagel (10% EtOAC in n-hexane). The compound was obtained as acolourless oil. (152 mg, 0.774 mmol, 77.4%).

¹H NMR (300 MHz, Chloroform-d) δ=4.07 (dtd, J=8.1, 7.0, 1.5, 4H), 3.14(d, J=2.3, 1H), 2.56 (tdd, J=7.0, 2.7, 0.6, 4H), 2.03 (t, J=2.7, 2H).¹³C NMR (75 MHz, Chloroform-d) δ=92.42 (d, J=1.3), 83.92 (d, J=47.1),80.24, 70.02, 65.81 (d, J=6.5), 21.28 (d, J=4.7). ³¹P NMR (122 MHz,CDCl₃) δ=130.15.

The procedures for the synthesis of compounds E3, E4 and E5 are providedherein below under “Procedures for the synthesis of compounds having acleavable group on the O-substituent”.

Staudinger Phosphonite Reaction with Phosphonite E1 and E2

The highly stable nature of electron rich phosphonites was furtherexploited by performing the Staudinger phosphonite reaction withalkyne-phosphonites in aqueous solvents. As depicted in FIG. 24 ,formation of the desired product from alkyne phosphonite E1 was observedin a pure aqueous system.

FIG. 24 shows: Reaction of an azido modified peptide with the watersoluble phosphonite E1 in Tris buffer. A: Reaction scheme. B:HPLC-trace; orange: starting material; blue: reaction after 2 h.

Procedures for the Staudinger Phosphonite Reaction with Phosphonites E1and E2 Peptide E9

Peptide E9 was synthesized by standard Fmoc-based chemistry in a linearsynthesis by manual coupling. 0.1 mmol of Rink amide resin (subst: 0.4mmol/g) was added to a reaction vessel and synthesis was performed withfive-fold amino acid excess. Fmoc de-blocking was achieved by resintreatment with 20% piperidine in DMF twice for 5 minutes. Coupling wasachieved by addition of HOBt/HBTU/DIPEA (5 eq./5 eq./10 eq) in DMF for45 min. After the final Gly coupling, 5 eq. of the 4-azido benzoic acidwas coupled with 5 eq. HATU and 10 eq. DIPEA in DMF for 45 min. Thepeptide was cleaved of the resin by addition of TFA/TIS/H₂O (95/2.5/2.5,w,w,w) within 3 h. Subsequently, the peptide was precipitated by theaddition of ice-cold diethyl ether. The precipitate was collected bycentrifugation, dried and purified by preparative HPLC (method Cdescribed above under “Procedures for the introduction of thealkyne-phosphonamidate moiety by generic building blocks via an amidebond”). ESI-MS for C37H50N₁₁O13⁺ [M+H]⁺ calcd: 856.36, found 856.36.

Staudinger Phosphonite Reaction of Peptide E9 with Amidate E1 in BasicTris-Buffer

10 μl of a 50 mM stock solution of peptide E9 in 100 mM Tris buffer (pH9.0) was added to 80 μl of 100 mM Tris buffer (pH 9.0). 10 μl of asolution of 500 mM phosphonite E1 in the same buffer was added andshaken at 37° C. for 2 hours at 800 RPM. A sample of 10 μl was drawn,diluted with 90 μl 1% TFA in H₂O and subjected to UPLC-MS-analysis.

Synthesis of E6

General procedure: 1.00 mmol of an organic azide (1.00 eq.) was stirredtogether with 1.00 mmol of an alkynyl phosphonite (1.00 eq.) in 5 ml DMFovernight. The organic solvent was removed under educed pressure and theresidue purified by column chromatography on silica. Following thisgeneral procedure, 37 mg Di-(3-Butinyl) ethynylphosphonite (compound E2)(0.192 mmol, 1.00 eq.) and 50 mg4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.162 mmol, 1.00 eq.)were mixed in 1 ml of DMF and purified by flash column chromatography onsilicagel (70% EtOAc in hexane). The compound was obtained as colourlessoil. (55 mg, 0.147 mmol, 76.6%).

¹H NMR (300 MHz, Chloroform-d) δ=8.33-7.93 (m, 3H), 7.21 (d, J=8.8, 2H),4.47-3.94 (m, 2H), 3.06 (d, J=13.2, 1H), 2.89 (s, 4H), 2.65 (td, J=6.7,2.7, 2H), 2.07 (t, J=2.7, 1H). ¹³C NMR (75 MHz, Chloroform-d) δ 169.61,161.42, 145.52, 132.30, 118.19, 117.72 (d, J=8.1 Hz), 89.38 (d, J=50.0Hz), 79.09, 70.94, 63.92 (d, J=5.0 Hz), 31.48, 25.69, 20.57 (d, J=8.2Hz). ³¹P NMR (122 MHz, CDCl₃) δ=−9.74.

Synthesis of Compounds Having a Cleavable Group on the O-Substituent andCleavage Experiments Introduction of a Cleavable Group on theO-Substituent

It has been described previously that cleavable disulfides can be usedto liberate a specific payload under reducing conditions. For example,this approach has been applied to the specific release of a cytotoxicpayload from an Antibody Drug Conjugate (ADC) within a cellularenvironment (31). In this context, it could be shown that disulfidesthat carry a leaving group in the beta position undergo cyclisation to athiirane after disulfide cleavage and liberate a given payload (32).

For the purpose of the present invention, the synthesis of a conjugatehaving a cleavable disulfide-comprising O-substituent was envisaged asshown in Scheme 29.

Scheme 29, which is depicted in FIG. 46 , shows the synthesis of aconjugate having a cleavable disulfide-comprising O-substituent via theStaudinger phosphonite reaction.

The following compounds having a cleavable group R on the O-substituentwere synthesized and subjected to cleavage experiments:

wherein R is

Procedures for the Synthesis of Compounds Having a Cleavable Group onthe O-Substituent

General Procedure 1 for the Synthesis of 2-Hydroxyethyl Disulfides

A 250 ml-round bottom flask was charged with 10 mmol (1.00 eq.) of therespective thiol, 10 mmol 2-mercaptoethanol (1.00 eq.), 0.1 mmol sodiumiodide and 20 ml EtOAc. The mixture was rapidly stirred and 10 mmol of asolution of 30% H₂O₂ in water was added drop-wise. The mixture wasstirred at room temperature for 1 h, volatiles were removed underreduced pressure and the disulfide was isolated by columnchromatography.

2-Hydroxyethyl Ethyldisulfide

The compound was synthesized according to the above “General procedure 1for the synthesis of 2-hydroxyethyl disulfides” from 2.00 ml Ethanethiol(27.74 mmol, 1.00 eq.), 1.96 ml 2-mercaptoethanol (27.74 mmol, 1.00eq.), 41 mg sodium iodide (0.28 mmol, 0.01 eq.) and 3.14 ml hydrogenperoxide solution (aqueous, 30%) (27.74 mmol, 1.00 eq.). The disulfidewas isolated by column chromatography on silica (20% EtOAc in hexane) ascolourless oil. Yield: 2.15 g (15.53 mmol, 56.0%).

¹H NMR (300 MHz, Chloroform-d) δ=3.91 (dd, J=5.7, 2H), 2.87 (t, J=5.7,2H), 2.74 (q, J=7.3, 2H), 2.08 (s, 1H), 1.35 (t, J=7.3, 3H).

2-Hydroxyethyl Isopropyldisulfide

The compound was synthesized according to the above “General procedure 1for the synthesis of 2-hydroxyethyl disulfides” from 2.00 mlisopropylthiol (21.53 mmol, 1.00 eq.), 1.52 ml 2-mercaptoethanol (21.53mmol, 1.00 eq.), 32 mg sodium iodide (0.21 mmol, 0.01 eq.) and 2.44 mlhydrogen peroxide solution (aqueous, 30%) (21.53 mmol, 1.00 eq.). Thedisulfide was isolated by column chromatography on silica (10% EtOAc inhexane) as colourless oil. Yield: 1.10 g (7.22 mmol, 33.6%)

¹H NMR (300 MHz, Chloroform-d) δ=3.88 (m, 2H), 3.02 (hept, J=6.7, 1H),2.85 (t, J=5.9, 2H), 2.37 (m, 1H), 1.32 (d, J=6.7, 6H). ¹³C NMR (75 MHz,CDCl₃) δ=60.48, 41.94, 41.14, 22.54.

2-Hydroxyethyl Tert-Butyldisulfide

The compound was synthesized according to the above “General procedure 1for the synthesis of 2-hydroxyethyl disulfides” from 2.00 mltert-Butylthiol (17.74 mmol, 1.00 eq.), 1.24 ml 2-mercaptoethanol (17.74mmol, 1.00 eq.), 26 mg sodium iodide (0.18 mmol, 0.01 eq.) and 2.04 mlhydrogen peroxide solution (aqueous, 30%) (17.74 mmol, 1.00 eq.). Thedisulfide was isolated by column chromatography on silica (10% EtOAc inhexane) as colourless oil. Yield: 0.90 g (5.41 mmol, 30.5%).

¹H NMR (300 MHz, Chloroform-d) δ=3.87 (t, J=5.9, 2H), 2.86 (t, J=5.9,2H), 2.33 (bs, 1H), 1.35 (s, 9H). NMR Data was in accordance withliterature values (33).

2-(3-Hydroxypropyl) Isopropyl Disulfide

A 500-ml round-bottom flask was charged with 2.00 ml thiolactic acid(23.55 mmol, 1.00 eq.) and 150 ml dry THF. At 0° C., 1.60 g Lithiumaluminium hydride (47.10, 2.0 eq.) were added portion-wise. The mixturewas stirred at room temperature for 1 h, cooled again to 0° C. andquenched carefully with 6 N HCl. The aqueous phase was extracted withtwice with 100 ml EtOAc, the organic fractions pooled, dried (MgSO₄) andall volatiles were removed under reduced pressure. The resultingcolourless oil was redissolved in 20 ml EtOH and 2.18 ml isobutyl thiol(23.55 mmol, 1.00 eq.), 55 mg sodium iodide (0.24 mmol, 0.01 eq.) and2.70 ml hydrogen peroxide solution (aqueous, 30%) (23.55 mmol, 1.00 eq.)were added. The yellowish solution was stirred for another hour.Volatiles were removed under reduced pressure and the above stateddisulfide isolated by column chromatography on silica (20% EtOAc inhexane) as colourless oil. Yield: 1.15 g (6.928 mmol, 29.4%).

¹H NMR (300 MHz, Chloroform-d) δ=3.69 (dd, J=5.8, 3.2, 2H), 3.08-2.83(m, 2H), 1.37-1.25 (m, 9H). ¹³C NMR (75 MHz, CDCl3) δ=65.49, 48.70,41.66, 22.60, 22.51, 16.89.

1-(4-(hydroxymethyl)phenyl)-2-phenyldiazene

The compound was synthesized according to previously published procedureand isolated as orange solid (34).

¹H NMR (300 MHz, Chloroform-d) δ=8.03-7.86 (m, 4H), 7.68-7.42 (m, 5H),4.81 (s, 2H). NMR Data was in accordance with literature values (34).

General Procedure 2 for the Synthesis of O-Substituted AlkynylPhosphonites from Bis(Diisopropylamino)Chlorophosphine

A 25-ml Schlenk flask was charged with 267 mgbis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.) under anargon atmosphere, cooled to 0° C. and 2.20 ml ethynylmagnesium bromidesolution (0.5 M in THF, 1.10 mmol, 1.10 eq.) was added drop wise. Theyellowish solution was allowed to warm to room temperature and stirredfor further 30 minutes. The respective alcohol, dissolved in 5.56 ml1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol) was added and thewhite suspension was stirred over night at room temperature. Thereaction mixture was directly placed on a silica gel flash column.

Di-(ethyl disulfido)ethyl) ethynylphosphonite

The compound was synthesized according to the above “General procedure 2for the synthesis of O-substituted alkynyl phosphonites frombis(diisopropylamino)chlorophosphine” from 116 mgbis(diisopropylamino)chlorophosphine (0.44 mmol, 1.00 eq.), 0.96 mlethynylmagnesium bromide solution (0.5 M in THF, 0.48 mmol, 1.10 eq.),150 mg 2-Hydroxyethyl ethyldisulfide (1.10 mmol, 2.50 eq.), 2.42 ml1H-tetrazole solution (0.45 M in MeCN, 1.10 mmol, 2.50 eq.) and purifiedby flash column chromatography on silicagel (10% to 20% EtOAc inhexane). The compound was obtained as yellowish oil. (112 mg, 0.34 mmol,77.0%).

¹H NMR (300 MHz, Chloroform-d) δ=4.22 (dt, J=7.6, 6.8, 4H), 3.15 (d,J=2.3, 1H), 2.95 (t, J=6.8, 4H), 2.75 (q, J=7.3, 4H), 1.35 (t, J=7.3,6H). ³¹P NMR (122 MHz, CDCl₃) δ=130.46.

Di-(2-isopropyl disulfido)ethyl) ethynylphosphonite

The compound was synthesized according to the above “General procedure 2for the synthesis of O-substituted alkynyl phosphonites frombis(diisopropylamino)chlorophosphine” from 213 mgbis(diisopropylamino)chlorophosphine (0.80 mmol, 1.00 eq.), 1.76 mlethynylmagnesium bromide solution (0.5 M in THF, 0.88 mmol, 1.10 eq.),370 mg 2-Hydroxyethyl isopropyldisulfide (2.00 mmol, 2.50 eq.), 4.44 ml1H-tetrazole solution (0.45 M in MeCN, 2.00 mmol, 2.50 eq.) and purifiedby flash column chromatography on silicagel (10% EtOAc in hexane). Thecompound was obtained as yellowish oil. (183 mg, 0.51 mmol, 63.9%).

¹H NMR (300 MHz, Chloroform-d) δ=4.21 (dt, J=8.0, 6.8, 4H), 3.15 (d,J=2.3, 1H), 3.04 (p, J=6.7, 2H), 2.94 (t, J=6.8, 4H), 1.33 (d, J=6.7,12H). ³¹P NMR (122 MHz, CDCl₃) δ=130.40.

Di-(2-tert-butyl disulfido)ethyl) ethynylphosphonite

The compound was synthesized according to the above “General procedure 2for the synthesis of O-substituted alkynyl phosphonites frombis(diisopropylamino)chlorophosphine” from 167 mgbis(diisopropylamino)chlorophosphine (0.63 mmol, 1.00 eq.), 1.38 mlethynylmagnesium bromide solution (0.5 M in THF, 0.69 mmol, 1.10 eq.),260 mg 2-Hydroxyethyl tert-butyldisulfide (1.57 mmol, 2.50 eq.), 3.48 ml1H-tetrazole solution (0.45 M in MeCN, 1.57 mmol, 2.50 eq.) and purifiedby flash column chromatography on silicagel (10% EtOAc in hexane). Thecompound was obtained as yellowish oil. (190 mg, 0.49 mmol, 78.5%).

¹H NMR (300 MHz, Chloroform-d) δ=4.20 (dt, J=7.9, 6.9, 4H), 3.14 (d,J=2.2, 1H), 2.95 (t, J=6.9, 4H), 1.36 (s, 18H). ¹³C NMR (75 MHz,Chloroform-d) δ=92.35 (d, J=1.0), 84.21 (d, J=47.8), 66.37 (d, J=6.1),47.98, 40.77 (d, J=4.3), 29.89. ³¹P NMR (122 MHz, CDCl₃) δ=130.28.

Di-((2-isopropyl disulfido)-3-propyl) ethynylphosphonite

The compound was synthesized according to the above “General procedure 2for the synthesis of O-substituted alkynyl phosphonites frombis(diisopropylamino)chlorophosphine” from 267 mgbis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 mlethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.),415 mg 2-(3-Hydroxypropyl) isopropyl disulfide (2.50 mmol, 2.50 eq.),5.55 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol, 2.50 eq.) andpurified by flash column chromatography on silicagel (0-10% EtOAc inhexane). The compound was obtained as a diastereomeric mixture asyellowish oil. (91 mg, 0.235 mmol, 23.5%).

¹H NMR (300 MHz, Chloroform-d) δ=4.28-4.06 (m, 2H), 3.99-3.81 (m, 2H),3.21-3.10 (m, 1H), 3.07-2.95 (m, 4H), 1.37-1.28 (m, 18H). ¹³C NMR (75MHz, Chloroform-d) δ=92.39 (d, J=3.6), 84.32 (d, J=49.3), 71.18 (d,J=4.9), 48.18-44.79 (m), 41.65, 22.55 (d, J=6.5), 17.12. ³¹P NMR (122MHz, CDCl₃) δ=130.56, 130.32, 130.10.

Di-(4-Acetoxy Benzyl) Ethynylphosphonite

The compound was synthesized according to the above “General procedure 2for the synthesis of O-substituted alkynyl phosphonites frombis(diisopropylamino)chlorophosphine” from 267 mgbis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 mlethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.),415 mg 2-(3-Hydroxypropyl) isopropyl disulfide (2.50 mmol, 2.50 eq.),5.55 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol, 2.50 eq.) andpurified by flash column chromatography on silicagel (30% EtOAc inhexane). The compound was obtained as a as colourless oil. (118 mg,0.306 mmol, 30.6%).

¹H NMR (300 MHz, Chloroform-d) δ=7.34 (d, J=8.5, 4H), 7.08 (d, J=8.5,4H), 4.95 (dd, J=8.4, 1.7, 4H), 3.20 (d, J=2.3, 1H), 2.32 (s, 6H). ¹³CNMR (75 MHz, Chloroform-d) δ=169.44, 150.37, 135.31 (d, J=4.3), 128.89,121.68, 92.68, 84.40 (d, J=47.6), 69.35 (d, J=6.8), 21.16. ³¹P NMR (122MHz, CDCl₃) δ=131.09.

Di (4-(diazophenyl)-benzyl) ethynylphosphonite

The compound was synthesized according to the above “General procedure 2for the synthesis of O-substituted alkynyl phosphonites frombis(diisopropylamino)chlorophosphine” from 98 mgbis(diisopropylamino)chlorophosphine (0.37 mmol, 1.00 eq.), 0.80 mlethynylmagnesium bromide solution (0.5 M in THF, 0.4 mmol, 1.10 eq.),195 mg 1-(4-(hydroxymethyl)phenyl)-2-phenyldiazene (0.93 mmol, 2.50eq.), 2.00 ml 1H-tetrazole solution (0.45 M in MeCN, 0.93 mmol, 2.50eq.) and purified by flash column chromatography on silicagel (0-10%EtOAc in hexane). The compound was obtained as orange solid. (82 mg,0.171 mmol, 46.3%).

¹H NMR (300 MHz, Chloroform-d) δ=7.98-7.86 (m, 8H), 7.59-7.44 (m, 10H),5.08 (d, J=8.5, 4H), 3.24 (d, J=2.3, 1H). ¹³C NMR (75 MHz, Chloroform-d)δ=152.60, 152.27, 140.55 (d, J=4.3), 131.07, 129.09, 128.24, 123.03,122.90, 92.89, 84.35 (d, J=47.2), 69.54 (d, J=6.9). ³¹P NMR (122 MHz,CDCl₃) δ=131.77.

General Procedure 3 for the Synthesis of O-Substituted AlkynylPhosphonamidates from Alkynyl Phosphonites and Azides

1.00 mmol of an organic azide (1.00 eq.) was stirred together with 1.00mmol of an alkynyl phosphonite (1.00 eq.) in 5 ml DMF overnight. Theorganic solvent was removed under educed pressure and the residuepurified by column chromatography on silica.

2-Isopropyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate

The compound was synthesized according to the above “General procedure 3for the synthesis of O-substituted alkynyl phosphonamidates from alkynylphosphonites and azides” from 147 mg Di-(2-isopropyl disulfido)ethyl)ethynylphosphonite (0.411 mmol, 1.00 eq.) and 106 mg4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.411 mmol, 1.00 eq.)and purified by flash column chromatography on silicagel (60% EtOAc inhexane). The compound was obtained as colourless oil. (80 mg, 0.175mmol, 42.6%).

¹H NMR (300 MHz, Chloroform-d) δ=8.08 (d, J=8.7, 2H), 7.20 (d, J=8.8,2H), 7.13 (d, J=7.5, 1H), 4.63-4.18 (m, 2H), 3.23-2.76 (m, 8H), 1.31(dd, J=6.7, 1.0, 6H). ³¹P NMR (122 MHz, CDCl₃) δ=−10.16.

2-tert-butyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate

The compound was synthesized according to the above “General procedure 3for the synthesis of O-substituted alkynyl phosphonamidates from alkynylphosphonites and azides” from 50 mg Di-(2-tert-butyl disulfido)ethyl)ethynylphosphonite (0.129 mmol, 1.00 eq.) and 33 mg4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.129 mmol, 1.00 eq.)and purified by flash column chromatography on silicagel (70% EtOAc inhexane). The compound was obtained as colourless solid. (29 mg, 0.0638mmol, 47.4%).

¹H NMR (300 MHz, Chloroform-d) δ=8.06 (d, J=8.7, 2H), 7.49 (d, J=7.5,1H), 7.21 (d, J=8.7, 2H), 4.58-4.26 (m, 2H), 3.09-2.81 (m, 7H), 1.33 (s,9H). ³¹P NMR (122 MHz, CDCl₃) δ=−9.98.

2-isopropyl disulfido-3-propyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate

The compound was synthesized according to the above “General procedure 3for the synthesis of O-substituted alkynyl phosphonamidates from alkynylphosphonites and azides” from 61 mg Di-((2-isopropyldisulfido)-3-propyl) ethynylphosphonite (0.158 mmol, 1.00 eq.) and 40 mg4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.158 mmol, 1.00 eq.)and purified by flash column chromatography on silicagel (70% EtOAc inhexane). The compound was obtained as mixture of diastereomers ascolourless oil. (32 mg, 0.068 mmol, 43.0%).

¹H NMR (300 MHz, Chloroform-d) δ=8.05 (d, J=8.7, 2H), 7.84-7.74 (m, 1H),7.21 (d, J=8.8, 2H), 4.57-4.27 (m, 2H), 3.20-2.66 (m, 7H), 1.46-1.21 (m,9H). ³¹P NMR (122 MHz, CDCl₃) δ=−9.87.

4-acetoxy-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynylphosphonamidate

The compound was synthesized according to the above “General procedure 3for the synthesis of O-substituted alkynyl phosphonamidates from alkynylphosphonites and azides” from 103 mg Di-(4-acetoxy benzyl)ethynylphosphonite (0.267 mmol, 1.00 eq.) and 69 mg4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.267 mmol, 1.00 eq.)and purified by flash column chromatography on silicagel (70% EtOAc inhexane). The compound was obtained as colourless oil. (36 mg, 0.077mmol, 28.7%).

¹H NMR (300 MHz, Chloroform-d) δ=7.53-7.34 (m, 2H), 7.20-6.99 (m, 7H),5.14 (d, J=8.8, 2H), 3.01 (d, J=13.3, 1H), 2.91 (s, 4H), 2.32 (s, 3H).³¹P NMR (122 MHz, CDCl₃) δ=−10.33.

4-Diazophenyl-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate

The compound was synthesized according to the above “General procedure 3for the synthesis of O-substituted alkynyl phosphonamidates from alkynylphosphonites and azides” from 71 mg Di (4-(diazophenyl)-benzyl)ethynylphosphonite (0.148 mmol, 1.00 eq.) and 39 mg4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.148 mmol, 1.00 eq.)and purified by flash column chromatography on silicagel (50% EtOAc inhexane). The compound was obtained as orange solid. (58 mg, 0.112 mmol,75.8%).

¹H NMR (600 MHz, DMSO-d₆) δ=9.44 (d, J=8.6, 1H), 8.02 (d, J=8.8, 2H),7.96-7.89 (m, 4H), 7.67 (d, J=8.5, 2H), 7.64-7.57 (m, 3H), 7.33 (d,J=8.8, 2H), 5.28 (ddd, J=45.1, 12.5, 8.7, 2H), 4.61 (d, J=13.0, 1H),2.88 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ=170.90, 161.75, 152.36,152.23, 147.36, 139.31 (d, J=7.6), 132.33, 132.17, 129.97, 129.41,123.14, 123.08, 118.17 (d, J=8.1), 117.25, 93.06 (d, J=46.9), 76.49 (d,J=265.4), 66.88, 25.98. ³¹P NMR (243 MHz, DMSO) δ=−10.42.

General Procedure 4 for the Amide Bond Formation BetweenPhosphonamidate-NHS Esters and EDANS

0.1 mmol NHS-phosphonamidate (1.00 eq.) and 0.12 mmol5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (1.20 eq.)were dissolved in 10 mL DMF. 0.40 mmol of DIPEA (4.0 eq.) was added andthe mixture stirred for 3 hours at room-temperature. All volatiles wereremoved under reduced pressure and the crude mixture was purified bypreparative HPLC using method E described above under “Procedures forthe introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”.

5-((2-(O-(2-Isopropyl-disulfido-ethyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonicacid

The compound was synthesized according to the above “General procedure 4for the amide bond formation between phosphonamidate-NHS esters andEDANS” from 72 mg2-Isopropyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate (0.157 mmol, 1.00 eq.), 54 mg5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.188 mmol,1.20 eq.) and 109 μl DIPEA (0.628 mmol, 4.0 eq.) and purified bysemi-preparative HPLC (method E described above under “Procedures forthe introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”). The compound was obtained as whitesolid. (62 mg, 0.102 mmol, 64.9%).

¹H NMR (600 MHz, DMSO-d₆) δ=8.90 (d, J=8.8, 1H), 8.61 (t, J=5.6, 1H),8.56 (d, J=8.6, 1H), 8.13 (d, J=8.3, 1H), 8.04 (dd, J=7.2, 1.1, 1H),7.82 (d, J=8.7, 2H), 7.60-7.31 (m, 2H), 7.17 (d, J=8.8, 2H), 4.51 (d,J=12.8, 1H), 4.37-4.17 (m, 2H), 3.65 (q, J=6.3, 2H), 3.52 (t, J=6.5,2H), 3.07 (p, J=6.7, 1H), 3.03 (t, J=6.3, 2H), 1.24 (dd, J=6.7, 2.8,6H). ¹³C NMR (151 MHz, DMSO-d₆) δ=167.03, 144.53, 143.31, 130.53,129.02, 127.63, 126.33, 125.44, 125.15, 124.70, 123.21, 117.55, 117.50,92.38 (d, J=45.7), 76.79 (d, J=262.5), 63.85 (d, J=4.7), 46.85, 40.77,39.01 (d, J=7.7), 37.65, 22.72. ³¹P NMR (243 MHz, DMSO) δ=−9.84.

5-((2-(O-(2-tert-butyl-disulfido-ethyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonicacid

The compound was synthesized according to the above “General procedure 4for the amide bond formation between phosphonamidate-NHS esters andEDANS” from 10 mg2-tert-butyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate (0.021 mmol, 1.00 eq.), 7 mg5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.025 mmol,1.20 eq.) and 15 μl DIPEA (0.084 mmol, 4.0 eq.) and purified bysemi-preparative HPLC (method E described above under “Procedures forthe introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”). The compound was obtained as whitesolid. (8 mg, 0.013 mmol, 62.3%).

¹H NMR (600 MHz, DMSO-d₆) δ=8.87 (d, J=8.7, 1H), 8.57 (t, J=5.8, 1H),8.21 (d, J=8.6, 1H), 8.10 (d, J=8.5, 1H), 7.94 (dd, J=7.1, 1.2, 1H),7.80 (d, J=8.7, 2H), 7.36 (dd, J=8.5, 7.1, 1H), 7.31 (dd, J=8.7, 7.5,1H), 7.14 (d, J=8.8, 2H), 6.74 (d, J=7.6, 1H), 4.49 (d, J=12.8, 1H),4.37-4.11 (m, 2H), 3.60 (q, J=6.4, 2H), 3.40 (t, J=6.5, 2H), 3.03 (t,J=6.5, 2H), 1.29 (s, 9H). ³¹P NMR (243 MHz, DMSO) δ=−9.87.

5-((2-(O-2-isopropyldisulfido-3-propyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonicacid

The compound was synthesized according to the above “General procedure 4for the amide bond formation between phosphonamidate-NHS esters andEDANS” from 29 mg 2-isopropyldisulfido-3-propyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate (0.061 mmol, 1.00 eq.), 21 mg5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.073 mmol,1.20 eq.) and 42 μl DIPEA (0.244 mmol, 4.0 eq.) and purified bysemi-preparative HPLC (method E described above under “Procedures forthe introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”). The compound was obtained as amixture of diastereomers as white solid. (15 mg, 0.024 mmol, 39.5%).

¹H NMR (600 MHz, DMSO-d₆) δ=8.88 (d, J=8.8, 1H), 8.58 (t, J=5.7, 1H),8.35 (d, J=8.6, 1H), 8.10 (dt, J=8.6, 1.1, 1H), 7.98 (dd, J=7.1, 1.1,1H), 7.81 (d, J=8.7, 2H), 7.39 (ddd, J=31.3, 8.6, 7.3, 2H), 7.15 (d,J=8.8, 2H), 6.91 (d, J=7.5, 1H), 4.51 (dd, J=12.9, 1.8, 1H), 4.25-4.13(m, 1H), 4.13-3.98 (m, 1H), 3.61 (q, J=6.4, 2H), 3.45 (t, J=6.6, 2H),3.18 (dtd, J=10.6, 6.8, 5.2, 1H), 3.03 (h, J=6.7, 1H), 1.30-1.19 (m,9H). ¹³C NMR (151 MHz, DMSO-d₆) δ=166.92, 144.74, 143.21, 130.61,128.96, 127.79, 126.43, 125.10, 124.61, 123.82, 123.09, 117.50 (d,J=7.5), 92.58 (d, J=9.5), 92.28 (d, J=9.4), 76.76 (d, J=262.3), 68.10(d, J=4.9), 45.48, 41.32 (d, J=8.6), 38.15, 22.74, 17.14 (d, J=4.1). ³¹PNMR (243 MHz, DMSO) δ=−9.76, −9.79.

5-((2-(O-(4-acetoxybenzyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonicacid

The compound was synthesized according to the above “General procedure 4for the amide bond formation between phosphonamidate-NHS esters andEDANS” from 36 mg4-acetoxy-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynylphosphonamidate (0.076 mmol, 1.00 eq.), 22 mg5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.095 mmol,1.20 eq.) and 53 μl DIPEA (0.284 mmol, 4.0 eq.) and purified bysemi-preparative HPLC (method E described above under “Procedures forthe introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”). The compound was obtained as whitesolid. (14 mg, 0.023 mmol, 30.4%).

¹H NMR (600 MHz, DMSO-d₆) δ=8.92 (d, J=8.6, 1H), 8.56 (t, J=5.7, 1H),8.32 (d, J=8.7, 1H), 8.10 (d, J=8.5, 1H), 8.03-7.92 (m, 1H), 7.80 (d,J=8.6, 2H), 7.47 (d, J=8.5, 2H), 7.41 (dd, J=8.5, 7.2, 1H), 7.36 (t,J=8.1, 1H), 7.17 (d, J=6.7, 2H), 7.15 (d, J=6.7, 2H), 6.88 (d, J=7.5,1H), 5.25-5.05 (m, 2H), 4.49 (d, J=12.8, 1H), 3.61 (q, J=6.3, 2H), 3.44(t, J=6.6, 2H), 2.28 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ=169.61,166.95, 150.96, 144.73, 143.29, 133.66 (d, J=7.7), 130.61, 129.81,128.99, 127.82, 126.47, 125.06, 124.53, 123.68, 123.10, 122.42, 117.44(d, J=7.9), 92.28 (d, J=45.6), 77.01 (d, J=261.8), 66.59 (d, J=4.4),45.26, 38.24, 21.31. ³¹P NMR (243 MHz, DMSO) δ=−9.87.

5-((2-(O-(4-Diazophenyl-benzyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonicacid

The compound was synthesized according to the above “General procedure 4for the amide bond formation between phosphonamidate-NHS esters andEDANS” from 27 mg4-Diazophenyl-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimideester)-P-ethynyl phosphonamidate (0.053 mmol, 1.00 eq.), 15 mg5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.064 mmol,1.20 eq.) and 37 μl DIPEA (0.212 mmol, 4.0 eq.) and purified bysemi-preparative HPLC (method E described above under “Procedures forthe introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”). The compound was obtained as orangesolid. (18 mg, 0.027 mmol, 50.9%).

¹H NMR (600 MHz, DMSO-d₆) δ=8.98 (d, J=8.7, 1H), 8.57 (t, J=5.7, 1H),8.36 (d, J=8.6, 1H), 8.11 (d, J=8.5, 1H), 7.98 (d, J=7.1, 1H), 7.97-7.88(m, 4H), 7.81 (d, J=8.8, 2H), 7.66 (d, J=8.5, 2H), 7.64-7.52 (m, 4H),7.42 (dd, J=8.5, 7.1, 1H), 7.37 (t, J=8.1, 1H), 7.18 (d, J=8.7, 2H),6.92 (d, J=7.5, 1H), 5.37-5.04 (m, 2H), 4.53 (d, J=12.8, 1H), 3.61 (q,J=6.4, 2H), 3.45 (t, J=6.6, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ=166.95,152.36, 152.18, 144.75, 143.27, 139.56, 139.51, 132.15, 130.61, 129.97,129.32, 129.01, 127.84, 126.44, 125.11, 124.62, 123.83, 123.13, 123.07,117.49 (d, J=8.0), 92.47 (d, J=45.9), 76.95 (d, J=262.7), 66.56 (d,J=4.4), 45.47, 38.15. ³¹P NMR (243 MHz, DMSO) δ=−9.68.

General Procedure 5 for the Addition of a Cys-Model Peptide to DifferentO-Substituted EDANS Phosphonamidates

The General procedure 5 for the addition of a Cys-model peptide todifferent O-substituted EDANS phosphonamidates is depicted in FIG. 47 .

Equal volumes of a 5 mM solution of the respective EDANS-phosphonamidatein DMF and a 5 mM solution of the above stated DABCYL-ModifiedCys-peptide in 100 mM NH₄HCO₃-Buffer (pH8.5) were freshly prepared,mixed and shaken at room temperature for 1 h. All volatiles were removedunder reduced pressure and the thiol adducts isolated bysemi-preparative HPLC (method E described above under “Procedures forthe introduction of the alkyne-phosphonamidate moiety by genericbuilding blocks via an amide bond”). Isolated conjugates were analyzedby HPLC-MS as set out in the following Table 5 and FIGS. 25A-25E:

TABLE 5 HPLC Isolated R trace Mass analysis Yield

see FIG. 25A C₇₄H₉₆N₁₅O₂₀PS₄ ²⁺ [M + 2H]²⁺ calcd: 836.78, found: 836.9439.4%

see FIG. 25B C₇₅H₉₈N₁₅O₂₀PS₄ ²⁺ [M + 2H]²⁺ calcd: 843.77, found: 844.1044.7%

see FIG. 25C C₇₅H₉₈N₁₅O₂₀PS₄ ²⁺ [M + 2H]²⁺ calcd: 843.77, found: 844.0524.5%

see FIG. 25D C₇₈H₉₄N₁₅O₂₂PS₂ ²⁺ [M + 2H]²⁺ calcd: 843.79, found: 844.1326.1%

see FIG. 25E C₈₂H₉₆N₁₇O₂₀PS₂ ²⁺ [M + 2H]²⁺ calcd: 866.81, found: 867.12Product not isolated

Procedure for the Cleavage of the Disulfide Containing Amidate-Adductswith TCEP

The procedure for the cleavage of the disulfide containingamidate-adducts with TCEP is depicted in FIG. 48 .

10 μl of a 1 mM stock solution of the respective peptide (SM1-3) inphosphate buffered saline (PBS) was premixed with 80 μl of PBS. 10 μl ofa 10 mM stock solution of Tris-(2-carboxyethyl)-phosphin (TCEP) in PBSwas added and the solutions were shaken at 37° C. for one hour. 15 μlsamples were drawn afterwards, diluted with 15 μl of 2% trifuloroaceticacid (TFA) solution in water and subjected to UPLC-MS analysis. TheUPLC-MS analysis is depicted in FIGS. 26A-26C. Red line shows incubationwith TCEP, black with PBS only. Peaks were identified by MS. The resultsshow that the disulfide-containing O-substituents are cleaved and theEDANS-containing part is liberated from the starting materials.

Procedure for the Cleavage of the Ester-Containing Amidate-Adducts withCell Lysate

The procedure for the cleavage of the ester-containing amidate-adductswith Cell lysate is depicted in FIG. 49 .

10 μl of a 1 mM stock solution of the peptide SM4 in PBS was premixedwith 90 μl of freshly prepared HeLa-lysate in PBS. The solutions wasshaken at 37° C. for one hour. A 15 μl sample was drawn afterwards,diluted with 15 μl of 2% TFA solution in water and subjected to UPLC-MSanalysis. The UPLC-MS analysis is depicted in FIG. 27 . Red line showsincubation with cell lysate, black with PBS only. Peaks were identifiedby MS. The results show that the O-substituent on the phosphoruscomprising an ester moiety is cleaved and the EDANS-containing part isliberated from the starting material.

Procedure for the Diazo-Containing Amidate-Adducts with SodiumDithionite

The procedure for the cleavage of the diazo-containing amidate-adductswith sodium dithionite is depicted in FIG. 50 .

10 μl of a 1 mM stock solution of the peptide SM5 in PBS was premixedwith 80 μl of PBS. 10 μl of a 200 mM stock solution of TCEP in PBS wasadded and the solutions were shaken at 37° C. for one hour. 15 μlsamples were drawn afterwards, diluted with 15 μl of 2% TFA solution inwater and subjected to UPLC-MS analysis. The UPLC-MS analysis isdepicted in FIG. 28 . Red line shows incubation with TCEP, black withPBS only. Peaks were identified by MS. The results show that theO-substituent on the phosphorus comprising a diazo moiety is cleaved andthe EDANS-containing part is liberated from the starting material.

Thus, it has been demonstrated that a cleavage of the amidates havingvarious cleavable groups as O substituent on the phosphorus is possible.

Without wishing to be bound by any theory, for a disulfide-containinggroup on the phosphorus it is believed that the mechanism of thecleavage proceeds as exemplarily depicted in Scheme 31 (FIG. 52 ), i.e.through reductive cleavage of the disulfide, cyclisation to a thiiraneto generate a free phosphonamidic acid which undergoes P—N-hydrolysis toliberate the payload as a free amine.

Scheme 30, which is depicted in FIG. 51 , shows the reductive cleavageand elimination mechanism.

Disulfide Substituted Phosphonites for Protein Conjugation

The cyclic cell-penetrating peptide c(Tat) was conjugated to eGFP viathe Staudinger induced thiol addition with a disulfide substitutedphosphonite.

First, we synthesized the cyclic Tat-peptide via solid phase peptidesynthesis (SPPS) (see Scheme 32). By capping the N-terminus with4-azidobenzoic acid we obtained compound E11 having an azide moiety.After purification by preparative HPLC the Staudinger phosphonitereaction of E11 with the disulfide containing alkyne phosphonites wascarried out in DMF to give compounds E12 and E13, which were purifiedagain by preparative HPLC.

Scheme 31, which is depicted in FIG. 52 , shows the SPPS of alkynefunctionalized cyclic Tat.

With the alkyne functionalized peptides in hand we further tested thethiol addition towards a cysteine containing eGFP as shown in FIG. 29 .The eGFP C70M S147C is a mutant, which exhibits only two cysteines ofwhich only one is addressable.

For the tert-butyl-disulfide substituent (E13) the thiol additionreaction went to completion after incubating eGFP with 6 equivalentsphosphonite in PBS at 37° C. for 16 hours at a proteinconcentration of63 μM. When applying the same reaction conditions with theisopropyl-disulfide substituent (E12) the product was obtained in about50% conversion according to MALDI analysis as shown in FIG. 29 .

FIG. 29 shows: Thiol addition of alkyne-c(Tat) to eGFP C70M S147C.

Procedures for the Disulfide Substituted Phosphonites for ProteinConjugation Synthesis of c(Tat)-azide

The c(Tat) was synthesized in a 0.1 mmol scale on a Rink Amide Resinwith a loading of 0.78 mm/g. The synthesis was carried out on a PTIsynthesizer with single couplings of each amino acid (10 eq. amino acidfor 40 min) in DMF. After the final building block coupling the peptide,still Fmoc protected, was treated with Pd(PPh₃)₄ (24 mg, 20 μmol, 20 mol%) and Phenylsilane (308 μl, 2.5 mmol, 2.5 eq.) in 4 ml dry DCM for 1hour in order to cleave the alloc and allyl protecting groups in onestep. After confirmation of full deprotection by test cleavage,cyclization with 2 eq. HATU 4 eq. DIPEA was carried out over night inDMF.

The peptide was then Fmoc-deprotected using 20% Piperidine in DMF andthe 4-azidobenzoic acid (81.6 mg, 0.5 mmol, 5 eq.) was coupled to theN-terminus with HATU (190.1 mg, 0.5 mmol, 5 eq.) and DIPEA (170 μl, 1.0mmol, 10 eq.) for 1 hour. Finally the peptide was cleaved from the resinby treatment with 4 ml of a TFA:TIS:H₂O (95:2.5:2.5) for 3 hours andprecipitated in cold diethylether. The crude peptide was purified bypreparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN(0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). Theproduct was gained as white powder (30.0 mg, 11.4 μmol, 11.4% yield) andwas analyzed by analytical UPLC (5 to 95% of acetonitrile in watercontaining 0.1% TFA on a RP-C18 column). LRMS: m/z: 648.49 [M+3H]³⁺(calcd. m/z: 648.0569).

Synthesis of c(Tat)-Phosphonamidate Alkyne: Staudinger Reaction onc(Tat)-azide

The purified c(Tat)-azido peptide (5 mg, 1.9 μmol, 1 eq.) was reactedwith both disulfide substituted phosphonites according to the generalprotocol. The crude peptide was purified by preparative reverse phaseC18 HPLC. The product was gained as white powder and was analyzed byMALDI-TOF.

Hydrothiolation of Electron-Deficient c(Tat)-Phosphonamidate Alkyne:Reaction with GFP C70M S147C

The hydrothiolation reaction of the electron-deficientc(Tat)-phosphonamidate alkyne with GFP C70M S147C is depicted in FIG. 53.

eGFP C70M S147C (2.7 nmol, 1 eq) in PBS was concentrated to 40 μl andc(Tat)-phosphonamidate alkyne (0.05 mg, 16.2 nmol, 6 eq.) was added.After the reaction mixture was shaken at 37° C. and 800 rpm over nightit was purified by ZebaSpin filters with a MWCO of 7 kDa. The productwas analyzed by MALDI-TOF. For the conjugation of peptide E12 anapproximately 50% conversion to the product was observed, while incontrast the conjugation of peptide E13 gave a full conversion.

MALDI TOF for E14: expected Product (in Da): 29919 (M+H⁺), 14960(M+2H⁺); found (in Da): 29933 (M+H⁺), 14967 (M+2H⁺)

MALDI TOF for E15: expected Product (in Da): 29933 (M+H⁺), 14967(M+2H⁺); found (in Da): 29940 (M+H⁺), 14965 (M+2H⁺)

Intramolecular Staudinger Induced Thiol Addition for Peptide Cyclization

The incorporation of an azide as well as a thiol into a complexmolecule, e.g. a peptide, leads the way for the intramolecularstaudinger induced thiol addition, that can realize an intramolecularcyclization as shown in FIG. 38 .

Without wishing to be bound by any theory, it is assumed that first theazide is reacting with the electron-rich alkyne/alkene-phosphonite uponwhich the phosphonamidate is formed and an electron-pooralkyne/alkene-phosphonamidate is formed that undergoes a fastintramolecular thiol addition with the cysteine in the peptidestructure.

First we synthesized a peptide taken from the protein sequence of BCL-9and we incorporated an azidohomoalanine and a cysteine distanced bythree amino acids into the peptide by standard solid phase peptidesynthesis. After cleavage from the solid phase and purification bypreparative HPLC we gained the peptide. With this in hand we could probethe intramolecular cyclization by staudinger induced thiol addition.

We reacted the in dry DMSO solubilized peptide with eitherdiethyl-ethynylphosphonite or diethyl-vinylphosphonite for 24 hours.After preparative HPLC the cyclized peptide was gained, which wasconfirmed by Ellman's test.

Procedures for the Intramolecular Staudinger Induced Thiol Addition forPeptide Cyclization Synthesis of BCL9-azide

The structure of the BCL9-azide is depicted in FIG. 54 .

The BCL9-azide was synthesized in a 0.1 mmol scale on a Rink Amide Resinwith a loading of 0.78 mm/g. The synthesis was carried out on a PTIsynthesizer with single couplings of each amino acid (5 eq. amino acidfor 40 min) in DMF. Finally the peptide was cleaved from the resin bytreatment with 4 ml of a TFA:TIS:H₂O (95:2.5:2.5) for 2 hours andprecipitated in cold diethylether. The crude peptide was purified bypreparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN(0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). Theproduct was gained as white powder (35.0 mg, 11.5 μmol, 11.5% yield) andwas analyzed by analytical UPLC (5 to 95% of acetonitrile in watercontaining 0.1% TFA on a RP-C18 column). LRMS: m/z: [M+3H]³⁺ 759.86(calcd. m/z: 759.6590).

Intramolecular Staudinger Induced Thiol Addition Alkyne-Phosphonamidate

The Staudinger reaction of the BCL-9azide with an alkyne-phosphonamidateis depicted in FIG. 55 .

Staudinger Reaction on BCL9-azide

The peptide 1 (20 mg, 6.55 μmol, 1 eq.) was dissolved in dry DMSO (1.5ml, 4.4 mM). After drying under high vacuum in a previously flame driedflask the Bisethoxyalkyne-phosphonite was given to the reaction mixture(volume according to percentage of product determined by NMR, 39.3 μmol,6 eq.). The reaction mixture was heated to 50° C. and stirred for 24hours. After addition of water, the reaction mixture was purified viabasic (10 mM ammonium acetate buffer pH 9.0/MeCN) semi-preparativereverse phase C18 Nucleodur HPLC (0-5 min 95/5, Buffer/MeCN; 5-70 min10/90, Buffer/MeCN) and gave the cyclized product as a white powder(3.82 mg, 1.22 μmol, 18.7% overall yield). The product was furtheranalyzed with an Ellman's test which showed that 97% of the cysteine wasreacted. The final product 2 was analyzed by LC-UV: rt. 5.0 min (0-1 min95/5, water (0.1% TFA)/MeCN (0.1% TFA); 1-16.5 min 5/95, water (0.1%TFA)/MeCN (0.1% TFA) on RP-C18 column) and mass. LRMS: m/z: [M+3H]³⁺1049.19 (calcd. m/z: 1048.5349).

Alkene-Phosphonamidate

The Staudinger reaction of the BCL-9azide with an alkene-phosphonamidateis depicted in FIG. 56 .

Staudinger Reaction on BCL9-azide

The peptide 3 (34 mg, 11.55 μmol, 1 eq.) was dissolved in dry DMSO (4ml, 2.9 mM). After drying under high vacuum in a previously flame driedflask the Bisethoxyvinyl-phosphonite was given to the reaction mixture(volume according to percentage of product determined by NMR, 39.3 μmol,6 eq.). The reaction mixture stirred for 24 hours at room temperature.After addition of water, the reaction mixture was purified bypreparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN(0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). Theproduct was gained as white powder (14.9 mg, 4.8 μmol, 41.3% yield) andwas analyzed by analytical UPLC (5 to 95% of acetonitrile in watercontaining 0.1% TFA on a RP-C18 column). LRMS: m/z: [M+4H]⁴⁺ 782.89(calcd. m/z: 782.6660). The product 4 was further analyzed with anEllman's test which showed that 99% of the cysteine was reacted.

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The invention claimed is:
 1. A compound of formula (V)

wherein:

represents a triple bond; X represents R₃—C; R₁ independently representsan optionally substituted aliphatic or aromatic residue; optionally, R₁represents C₁-C₈-alkyl optionally substituted with at least one of(C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, Br, I, —NO₂,—N(C₁-C₈-alkyl)H, —NH₂, —N(C₁-C₈-alkyl)₂, ═O, C₃-C₈-cycloalkyl,—S—S—(C₁-C₈-alkyl), hydroxy-(C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4,5 or 6, C₂-C₈-alkynyl or optionally substituted phenyl; or optionally,R₁ represents phenyl optionally independently substituted with at leastone of C₁-C₈-alkyl, (C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5 or 6,F, Cl, I, Br, —NO₂, —N(C₁-C₈-alkyl)H, —NH₂ or —N(C₁-C₈-alkyl)₂; oroptionally, R₁ represents a 5- or 6-membered heteroaromatic system; R₃represents H or C₁-C₈-alkyl; and ● represents an aliphatic or aromaticresidue.
 2. The compound according to claim 1, wherein R₁ independentlyrepresents methyl, ethyl, propyl or butyl.
 3. The compound according toclaim 1, wherein R₁ represents

wherein R₁₀ and R₁₁ independently represent hydrogen or C₁-C₈-alkyl; and# represents the position of O.
 4. The compound according to claim 1,wherein R₁ represents C₁-C₈-alkyl substituted with phenyl, said phenylbeing further substituted with

wherein Z is O or NH, and wherein # represents the position of saidphenyl.
 5. The compound according to claim 1, wherein R₁ representshydroxyethyl or homopropargyl.
 6. The compound according to claim 1,wherein R₁ represents C₁-C₈-alkyl substituted with (C₁-C₈-alkoxy)_(n)wherein n is 1, 2, 3, 4, 5 or
 6. 7. The compound according to claim 1,wherein R₁ represents C₁-C₈-alkyl substituted withhydroxy-(C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5 or
 6. 8. Thecompound according to claim 1, wherein R₁ represents—(CH₂)₂—O—(CH₂)₂—OH.
 9. The compound according to claim 1, wherein ●represents an optionally substituted C₁-C₈-alkyl; an optionallysubstituted phenyl; or an optionally substituted 5- or 6-memberedheteroaromatic system.
 10. The compound according to claim 1, wherein ●represents a radioactive or non-radioactive nuclide, biotin, a reporterenzyme, a nucleotide, an oligonucleotide, a fluorophore, an amino acid,or a peptide.
 11. The compound according to claim 1, wherein ●represents a linker, a drug, or a linker-drug conjugate.
 12. A processfor the preparation of a compound of formula (V), said processcomprising the steps of: (I) reacting compound of formula (III)

wherein:

represents a double bond or a triple bond; X represents R₃—C when

is a triple bond; or X represents CR₃(R₄) when

is a double bond; R₁ independently represents an optionally substitutedaliphatic or aromatic residue; optionally, R₁ represents C₁-C₈-alkyloptionally substituted with at least one of (C₁-C₈-alkoxy)_(n) wherein nis 1, 2, 3, 4, 5 or 6, F, Cl, Br, I, —NO₂, —N(C₁-C₈-alkyl)H, —NH₂,—N(C₁-C₈-alkyl)₂, ═O, C₃-C₈-cycloalkyl, —S—S—(C₁-C₈-alkyl),hydroxy-(C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5 or 6,C₂-C₈-alkynyl or optionally substituted phenyl; or optionally, R₁represents phenyl optionally independently substituted with at least oneof C₁-C₈-alkyl, (C₁-C₈-alkoxy)_(n) wherein n is 1, 2, 3, 4, 5 or 6, F,Cl, I, Br, —NO₂, —N(C₁-C₈-alkyl)H, —NH₂ or —N(C₁-C₈-alkyl)₂; oroptionally, R₁ represents a 5- or 6-membered heteroaromatic system; R₃represents H or C₁-C₈-alkyl; and R₄ represents H or C₁-C₈-alkyl; with anazide of formula (IV)

wherein ● represents an aliphatic or aromatic residue; to prepare acompound of formula (V)

wherein ●,

, R₁, and X are as defined above.
 13. The compound according to claim 1,wherein R₁ represents ethyl.
 14. The compound according to claim 1,wherein R₁ represents an aliphatic residue, wherein the aliphaticresidue is a polymer.
 15. The compound according to claim 1, wherein R₁represents an aliphatic residue, wherein the aliphatic residue is apolyethyleneglycol.
 16. The compound according to claim 1, wherein ●represents an optionally substituted phenyl.
 17. The compound accordingto claim 1, wherein ● represents a linker-drug conjugate.
 18. Theprocess according to claim 12, wherein R₁ represents ethyl.
 19. Theprocess according to claim 12, wherein R₁ represents hydroxyethyl orhomopropargyl.
 20. The process according to claim 12, wherein R₁represents C₁-C₈-alkyl substituted with (C₁-C₈-alkoxy)_(n) wherein n is1, 2, 3, 4, 5 or
 6. 21. The process according to claim 12, wherein R₁represents C₁-C₈-alkyl substituted with hydroxy-(C₁-C₈-alkoxy)_(n)wherein n is 1, 2, 3, 4, 5 or
 6. 22. The process according to claim 12,wherein R₁ represents —(CH₂)₂—O—(CH₂)₂—OH.
 23. The process according toclaim 12, wherein R₁ represents an aliphatic residue, wherein thealiphatic residue is a polymer.
 24. The process according to claim 12,wherein R₁ represents an aliphatic residue, wherein the aliphaticresidue is a polyethyleneglycol.
 25. The process according to claim 12,wherein ● represents an optionally substituted phenyl.
 26. The processaccording to claim 12, wherein ● represents a linker-drug conjugate.